Weiping Li

A novel HDAC6 inhibitor Tubastatin A: Controls HDAC6-p97/VCP-mediated ubiquitination-autophagy turnover and reverses Temozolomide-induced ER stress-tolerance in GBM cells

Temozolomide (TMZ) is the cornerstone of therapy for glioblastoma multiforme (GBM). However, its efficacy is limited due to the development of multidrug resistance (MDR). In this study, we first identified the occurrence of ER stress-tolerance (ERST) in glioma cells and confirmed that ERST was positively correlated with TMZ resistance. We further showed that the seesaw-effect of HDAC6-p97/VCP (increased HDAC6 and decreased p97/VCP) in glioma cells was crucial to ERST-associated TMZ resistance. Moreover, the combination treatment of Tubastatin A (TUB, a selective inhibitor of HDAC6) and TMZ synergistically overcame ERST, reduced cell viability and induced apoptosis in TMZ-resistant glioma cells. TUB and TMZ triggered pro-apoptotic signals of the unfolded protein response (UPR) and ER stress and reversed the ratio between HDAC6 and p97/VCP, which potentially attenuated the activation of heat shock proteins and mediated the reversal of ERST. The combination treatment also triggered the dissociation of Dynein-HDAC6 and attenuation of the Dynein-Dynactin motor complex. In addition, this treatment induced HDAC6-p97/VCP-mediated ubiquitination-autophagy turnover, which was involved in the degradation and clearance of ubiquitinated misfolded proteins. This effect could be partially reversed by HDAC6 KO and/or p97/VCP overexpression. Therefore, we proposed that glioma cells optimized the clearance of ubiquitinated misfolded proteins via the reinforcement of HDAC6-facilitated autophagy and attenuation of the p97/VCP-mediated ubiquitin-proteasome system (UPS). In conclusion, our findings showed that the balance of HDAC6-p97/VCP was crucial to ERST-associated TMZ resistance and that HDAC6 inhibition might be a synergistic target and strategy along with TMZ for the improvement of clinical glioma treatment.

HPOB, an HDAC6 inhibitor, attenuates corticosterone-induced injury in rat adrenal pheochromocytoma PC12 cells by inhibiting mitochondrial GR translocation and the intrinsic apoptosis pathway

High levels of glucocorticoids (GCs) have been reported to damage normal hippocampal neurons, and such damage has been positively correlated with major depression (MD) and chronic stress. Our previous study showed that HDAC6 might be a potential target to regulate GC-induced glucocorticoid receptor (GR) translocation to the mitochondria and subsequent apoptosis. In the present study, we investigated the effect of HPOB, a selective HDAC6 inhibitor, on corticosterone (Cort)-induced apoptosis and explored the possible mechanism of action of HPOB in rat adrenal pheochromocytoma (PC12) cells, which possesses typical neuron features and expresses high levels of glucocorticoid receptors. We demonstrated that pre-treatment with HPOB remarkably reduced Cort-induced cytotoxicity and confirmed the anti-apoptotic effect of HPOB via the caspase-3 activity assay and H33342/PI and TUNEL double staining. Mechanistically, we demonstrated that HPOB reversed the Cort-induced elevation of GR levels in the mitochondria and blocked concomitant mitochondrial dysfunction and the intrinsic apoptosis pathway. Furthermore, HPOB was shown to attenuate expression of the multi-chaperone machinery (Hsp90-Hop-Hsp70) and cooperate with mitochondrial translocase of the outer/inner membrane (TOM/TIM) complex recruitment by triggering hyperacetylation of Hsps through HDAC6 inhibition. Considering all of these findings, the neuroprotective effect of HPOB demonstrated the crucial role of HDAC6 inhibition in reducing Cort-induced apoptosis in PC12xa0cells. The data further suggested that the anti-apoptotic activity of HDAC6 inhibition against the mitochondria-mediated impairment pathway might be mechanistically linked to the hyperacetylation of Hsps and consequent suppression of GR translocation to the mitochondria.

Radicol, a Novel Trinorguaiane-Type Sesquiterpene, Induces Temozolomide-Resistant Glioma Cell Apoptosis via ER Stress and Akt/mTOR Pathway Blockade

Glioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system (CNS) in adults. Multidrug resistance (MDR) results in undesirable prognosis during GBM chemotherapy. In this study, we determined that Radicol (RAD), a novel trinorguaiane‐type sesquiterpene originally isolated from the root of Dictamnus radicis Cortex, exhibited potently cytotoxic effect on temozolomide (TMZ)‐resistant GBM cell lines in a dose‐dependent manner. Radicol‐induced apoptosis was confirmed with Hoechst 33342/propidium iodide and terminal deoxynucleotidyl transferase‐mediated biotinylated UTP nick end‐labelling (TUNEL) staining. Studies investigating the mechanism revealed that RAD triggered an attenuation of protein disulphide isomerase (PDI) and induced the unmitigated unfolded protein response (UPR) and lethal endoplasmic reticulum (ER) stress. Simultaneously, we further demonstrated that RAD suppressed the activation of Akt/mTOR/p70S6K phosphorylation by up‐regulating the induction of glycogen synthase kinase‐3β (GSK‐3β). These results established a link between RAD‐induced ER stress and inhibition of the Akt/mTOR/p70S6K pathway, and the attenuation of PDI and activation of GSK‐3β might be the synergistic target of antineoplastic effects during RAD‐induced apoptosis. These findings suggested that RAD, possessing multiple cytotoxicity targets, low molecular weight and high lipid solubility, could be a promising agent for the treatment of malignant gliomas. Copyright

Tanshinone IIA induces apoptosis via inhibition of Wnt/β‑catenin/MGMT signaling in AtT‑20 cells

A strategy to suppress the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) by inhibition of Wnt/β-catenin signaling may be useful as a novel treatment for pituitary adenoma. Previous studies have reported that Tanshinone IIA (TSA), a major quinone compound isolated from Salvia miltiorrhiza, had antitumor effects. However, whether TSA has antitumor effects against pituitary adenoma and whether the mechanisms are associated with the Wnt/β-catenin/MGMT pathway remains to be clarified. In the present study, TSA treatment caused apoptosis in AtT-20 cells in a concentration-dependent manner, as demonstrated by cell viability reduction, phophatidylserine externalization detected by Annexin V staining and mitochondrial membrane potential disruption detected by JC-1 staining, which were associated with activation of caspase-3 and DNA fragmentation detected by TUNEL in AtT-20 cells. T-cell factor (TCF)-lymphoid-enhancing factor (LEF) reporter activity was determined by dual luciferase reporter assay and the interaction between β-catenin and TCF-4 were detected using a co-immunoprecipitation kit. The results indicated TSA treatment increased β-catenin phosphorylation, inhibited β-catenin nuclear translocation, reduced β-catenin/TCF-4 complex formation and TCF-LEF luciferase reporter activity, and subsequently reduced the expression of cyclin D1 and MGMT. Notably, overexpression of MGMT in β-catenin knock down AtT-20 cells abrogated the TSA-mediated effects in AtT-20 cells. In conclusion, TSA induced apoptosis via inhibition of Wnt/β-catenin-dependent MGMT expression, which may provide novel insights into the understanding of the mechanism of the antitumor effects of Salvia miltiorrhiza.

High expression of immunity-related GTPase family M protein in glioma promotes cell proliferation and autophagy protein expression

Glioma is the commonest malignant tumor in the central nervous system (CNS), characterized by rapid growth. However, the molecular mechanism underlying the growth remains unclear. Immunity-related GTPase family M protein (IRGM) participates in immune response to pathogen and tumorigenesis. Proliferation and autophagy are two crucial functions contributing to aggressive growth. Therefore, our aims were to probe whether IRGM regulates glioma proliferation and autophagy. In this study, we found that 47 glioma specimens had more IRGM expression than 11 non-cancerous brain tissues with immunohistochemistry. IRGM was also up-regulated in human glioma cell lines U87, U251 and A172 and so on compared with immortalized astrocytes. Importantly, overexpression of IRGM significantly increased the cell colonies formation, cell proliferation and Akt activation (Thr308 and Ser473 sites) than matched control. On another hand, all of IRGM, autophagy marker LC3II and autophagy adaptor p62 gradually increased after starvation 2 and 4u2009h. Furthermore, western blot and immunofluorescence results showed that knockdown of IRGM inhibited the formation of LC3-II and the expression of p62. Our data uncovered that IRGM acted in glioma proliferation and autophagy, providing a new target with dual roles for the future translation research.

Mechanisms Underlying H 2 O 2 -Evoked Carbonyl Modification of Cytoskeletal Protein and Axon Injury in PC-12 Cells

Background/Aims: To investigate the mechanism that enables oxidative stress and cytoskeleton protein carbonylation to contribute to axonal dysfunction in traumatic brain injury (TBI). Methods: We created an in vitro model of neuronal oxidative damage by exposing a neuron-like cell line (PC-12) to different concentrations (100 μM, 200 μM, and 300 μM) of H2O2 for 24 h or 48 h. Carbonyl modification of cytoskeletal proteins (β-actin and β-tubulin) and its impact on β-actin/β-tubulin filament dynamics were determined by enzyme-linked immunosorbent assay, immunostaining, and western blotting. Depolymerization of β-actin/β-tubulin filaments was evaluated using the monomer/polymer ratio of each protein via western blotting. Phosphorylation of the neurofilament heavy chain (P-NFH) was used as an axonal injury marker and detected by immunostaining. Results: Our results showed that H2O2 treatment led to increased oxidative stress in PC-12 cells, as indicated by the increased generation of malondialdehyde and 8-hydroxydeoxyguanosine and decreased intracellular glutathione levels. H2O2 treatment also increased carbonyl modification of total proteins and cytoskeleton proteins β-actin/β-tubulin, which occurred concurrently with the suppression of proteasome activity. Moreover, H2O2 treatment increased the generation of the axonal injury marker P-NFH, and depolymerization of the β-actin/β-tubulin filaments was indicated by increased monomer/polymer ratios of each protein. Lastly, overexpression of the proteasome β5 subunit in PC-12 cells significantly reduced the H2O2-induced accumulation of carbonylated β-actin/ β-tubulin, P-NFH, and β-actin/β-tubulin depolymerization. Conclusions: We concluded that carbonylation of cytoskeleton proteins could lead to depolymerization of their filaments and axonal injury, and proteasome suppression contributes to the accumulation of carbonylated proteins under oxidative conditions.

IRGM promotes glioma M2 macrophage polarization through p62/TRAF6/NF-κB pathway mediated IL-8 production: IRGM regulates GAM polarization

Alternatively activated (M2) macrophage promotes glioma progression and immune escape as the most immunocyte in glioma microenvironment. Finding out the key protein regulating M2 macrophage polarization is necessary for improving treatment. Whether immunity related GTPase M (IRGM) is involved in glioma development and M2 macrophage polarization is unknown. IRGM and M2 macrophage marker CD206 expression were examined using immunohistochemistry among 35 glioma and 11 non‐cancerous brain specimens. We found IRGM scores were positively correlated with CD206 scores in glioma specimens and monocyte proportion in blood samples. A172 glioma cells transfected with either IRGM knock‐down lentivirus (Lenti‐IRGM) or control lentivirus (Lenti‐HK) were subcutaneously injected into nude mice. In vivo, xenografted glioma size of the Lenti‐IRGM group was smaller and had weaker fluorescence signal than Lenti‐HK control group. Immunofluorescence results showed that there was obviously decreased IRGM, CD206, and IL‐8 expression in the mice glioma of Lenti‐IRGM group than Lenti‐HK control group. In vitro, flow cytometry results showed that M2 polarization from THP‐1 cocultured with Lenti‐IRGM glioma cells decreased in contrast to that with Lenti‐HK glioma cells; there were less interleukin‐8 (IL‐8) and macrophage inflammation protein 3‐α (MIP‐3α), but more interleukin‐6 (IL‐6) in the supernatant of Lenti‐IRGM glioma cells than matched control. Western blot and immunofluorescence displayed that IRGM strongly promoted sequestosome‐1 (p62/SQSTM1), necrosis factor receptor‐activating factor 6 (TRAF6) expression and NF‐κB transportation to the nucleus. Realtime PCR results demonstrated IRGM also promoted NF‐κB downstream cytokines IL‐8 and MIP‐3α mRNA expression. These data suggested that IRGM could promote glioma development and M2 macrophage polarization by regulating p62/TRAF6/NF‐κB pathway‐mediated IL‐8 production.

Hemangiopericytoma Arose From the Site of Meningioma Resection

Intracranial hemangiopericytoma (HPC) is a rare tumor and accounts for 0.4% of all primary central nervous system tumors. However, it has aggressive behavior in growth and infiltration. The importance of diagnosis and treatment between HPC and meningioma cannot be overemphasized. It is rare that HPC arises from the site of benign meningioma resection. And it would be benefitted to take HPC into account when coming across a patient with meningioma recurrence.

A Stratified Algorithm for Skull Base Reconstruction With Endoscopic Endonasal Approach

Abstract The authors presented our institutional experience with skull base reconstruction techniques and developed a stratified algorithm for different causes of cerebrospinal fluid (CSF) leak. This is a retrospective review of patients who were diagnosed as CSF leak treated with skull base reconstruction or who underwent endoscopic transsphenoidal surgery for sellar and parasellar lesions at our department from August 2012 to April 2017. The authors totally identified 57 (59 operations in total) patients who were divided into 3 groups according to the causes of CSF leak and different reconstruction techniques. All patients underwent skull base reconstruction with specific focus on diagnosis, reconstruction techniques and strategies, and clinical outcome. The reconstruction technique we adopted was the classical multiple-layer technique, as known as “sandwich” technique, with combination of fat tissue, septal bone, autologous fascia lata, artificial dura, and nasoseptal flap (NSF). The NSF was selectively harvested for large defects according to our protocol. The reconstruction failure rate is 4.4% (2 of 45) in patients underwent endoscopic surgery for sellar and parasellar lesions. Reconstruction for postoperative iatrogenic, traumatic, and spontaneous CSF leak achieved 100% success rate; 54.2% (32 of 59) operations were done with “sandwich” plus NSF. The overall failure rate of all reconstructions was 3.4% (2 of 59). A stratified approach with multiple-layer technique and NSF is reliable for skull base reconstruction.

Suspected Acoustic Neuroma Demyelinating Lesions.

Demyelinating lesions were recognized as a kind of rare central nervous system demyelinating lesion. The diagnosis and differential diagnosis of demyelinating lesions is difficult. Once the diagnosis was delayed or incorrect, it will make a great impact on patients.Demyelinating lesions often involved in young and middle-aged, but this patient was the aged, which is rare.