Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

Although the incidence of glioma is relatively low, it is the most malignant tumor of the central nervous system. The prognosis of high-grade glioma patient is very poor due to the difficulties in complete resection and resistance to radio-/chemotherapy. Therefore, it is worth investigating the molecular mechanisms involved in glioma drug resistance. MicroRNAs have been found to play important roles in tumor progression and drug resistance. Our previous work showed that miR-181b is involved in the regulation of temozolomide resistance. In the current study, we investigated whether miR-181b also plays a role in antagonizing the effect of teniposide. MiR-181b expression was measured in 90 glioma patient tissues and its relationship to prognosis of these patients was analyzed. Cell sensitivity to teniposide was tested in 48 primary cultured glioma samples. Then miR-181b stably overexpressed U87 cells were generated. The candidate genes of miR-181b from our previous study were reanalyzed, and the interaction between miR-181b and target gene MDM2 was confirmed by dual luciferase assay. Cell sensitivity to teniposide was detected on miR-181b over expressed and MDM2 down regulated cells. Our data confirmed the low expression levels of miR-181b in high-grade glioma tissues, which is related to teniposide resistance in primary cultured glioma cells. Overexpression of miR-181b increased glioma cell sensitivity to teniposide. Through target gene prediction, we found that MDM2 is a candidate target of miR-181b. MDM2 knockdown mimicked the sensitization effect of miR-181b. Further study revealed that miR-181b binds to the 3’-UTR region of MDM2 leading to the decrease in MDM2 levels and subsequent increase in teniposide sensitivity. Partial restoration of MDM2 attenuated the sensitivity enhancement by miR-181b. MiR-181b is an important positive regulator on glioma cell sensitivity to teniposide. It confers glioma cell sensitivity to teniposide through binding to the 3’-UTR region of MDM2 leading to its reduced expression. Our findings not only reveal the novel mechanism involved in teniposide resistance, but also shed light on the optimization of glioma treatment in the future.

Given that glioma stem cells (GSCs) play a critical role in the initiation and chemoresistance in glioblastoma multiforme (GBM), targeting GSCs is an attractive strategy to treat GBM. Utilizing an anti-cancer compound library, we identified R406, the active metabolite of a FDA-approved Syk inhibitor for immune thrombocytopenia (ITP), with remarkable cytotoxicity against GSCs but not normal neural stem cells. R406 significantly inhibited neurosphere formation and triggered apoptosis in GSCs. R406 induced a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) and subsequently production of excess ROS in GSCs. R406 also diminished tumor growth and efficiently sensitized gliomas to temozolomide in GSC-initiating xenograft mouse models. Mechanistically, the anti-GSC effect of R406 was due to the disruption of Syk/PI3K signaling in Syk-positive GSCs and PI3K/Akt pathway in Syk-negative GSCs respectively. Overall, these findings not only identify R406 as a promising GSC-targeting agent but also reveal the important role of Syk and PI3K pathways in the regulation of energy metabolism in GSCs.

Differentiation therapy using small molecules is a promising strategy for improving the prognosis of glioblastoma (GBM). Histone acetylation plays an important role in cell fate determination. Nevertheless, whether histone acetylation in specific sites determines GBM cells fate remains to be explored. Through screening from a 349 small molecule-library, we identified that histone deacetylase inhibitor (HDACi) MS-275 synergized with 8-CPT-cAMP was able to transdifferentiate U87MG GBM cells into neuron-like cells, which were characterized by cell cycle arrest, rich neuron biomarkers, and typical neuron electrophysiology. Intriguingly, acetylation tags of histone 3 at lysine 9 (H3K9ac) were decreased in the promoter of multiple oncogenes and cell cycle genes, while ones of H3K9ac and histone 3 at lysine 14 (H3K14ac) were increased in the promoter of neuron-specific genes. We then compiled a list of genes controlled by H3K9ac and H3K14ac, and proved that it is a good predictive power for pathologic grading and survival prediction. Moreover, cAMP agonist combined with HDACi also induced glioma stem cells (GSCs) to differentiate into neuron-like cells through the regulation of H3K9ac/K14ac, indicating that combined induction has the potential for recurrence-preventive application. Furthermore, the combination of cAMP activator plus HDACi significantly repressed the tumor growth in a subcutaneous GSC-derived tumor model, and temozolomide cooperated with the differentiation-inducing combination to prolong the survival in an orthotopic GSC-derived tumor model. These findings highlight epigenetic reprogramming through H3K9ac and H3K14ac as a novel approach for driving neuron-fate-induction of GBM cells.

Background: Gliomas represent the largest class of primary central nervous system neoplasms, many subtypes of which exhibit poor prognoses. Surgery followed by radiotherapy and chemotherapy has been used as a standard strategy but yielded unsatisfactory improvements in patient survival outcomes. The S-phase kinase protein 2 (Skp2), a critical component of the E3-ligase SCF complex, has been documented in tumorigenesis in various cancer types but its role in glioma has yet to be fully clarified. In this study, we investigated the function of Skp2 in the proliferation, stem cell maintenance, and drug sensitivity to temozolomide (TMZ) of glioma.Methods: To investigate the role of Skp2 in the prognosis of patients with glioma, we first analyzed data in databases TCGA and GTEx. To further clarify the effect of Skp2 on glioma cell proliferation, we suppressed its level in glioblastoma (GBM) cell lines through knockdown and small molecule inhibitors (lovastatin and SZL-P1-41). We then detected cell growth, colony formation, sphere formation, drug sensitivity, and in vivo tumor formation in xenograft mice model.Results: Skp2 mRNA level was higher in both low-grade glioma and GBM than normal brain tissues. The knockdown of Skp2 increased cell sensitivity to TMZ, decreased cell proliferation and tumorigenesis. In addition, Skp2 level was found increased upon stem cells enriching, while the knockdown of Skp2 led to reduced sphere numbers. Downregulation of Skp2 also induced senescence. Repurposing of lovastatin and novel compound SZL-P1-41 suppressed Skp2 effectively, and enhanced glioma cell sensitivity to TMZ in vitro and in vivo.Conclusion: Our data demonstrated that Skp2 modulated glioma cell proliferation in vitro and in vivo, stem cell maintenance, and cell sensitivity to TMZ, which indicated that Skp2 could be a potential target for long-term treatment.