Autophagy represents an alternative tumor-suppressing mechanism that overcomes the dramatic resistance of malignant gliomas to radiotherapy and proapoptotic-related chemotherapy. This study reports that valproic acid (VPA), a widely used anti-epilepsy drug, induces autophagy in glioma cells. Autophagy, crucial for VPA-induced cell death, is independent of apoptosis, even though apoptotic machinery is proficient. Oxidative stress induced by VPA occurs upstream of autophagy. Oxidative stress also activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereas blocking this pathway inhibits autophagy and induces apoptosis. VPA-induced autophagy cannot be alleviated by inositol, suggesting a mechanism different from that for lithium. Moreover, VPA potentiates autophagic cell death, but not apoptosis, when combined with other autophagy inducers such as rapamycin, Ly294002, and temozolomide in glioma cells both in vitro and in vivo, which may warrant further investigation toward possible clinical application in patients with malignant gliomas.

Overexpression of ERCC1 mRNA is associated with drug resistance to cisplatin in human gliomas, but the role of the ERCC1 promoter in drug resistance has not been demonstrated. We have used sodium bisulfite sequencing to compare ERCC1 promoter methylation patterns in cisplatin‐sensitive and cisplatin‐resistant glioma cells. The levels of ERCC1 DNA methylation, mRNA and protein in 32 human glioma samples were examined by methylation specific PCR, real‐time RT‐PCR and immunohistochemistry, respectively. Meanwhile, cisplatin sensitivities to these human glioma samples were tested by histoculture drug response assay. Hypermethylation was observed in the upstream 5Kb region of the ERCC1 promoter of cisplatin‐sensitive glioma cell lines. ERCC1 DNA methylation levels were highly variable in 32 human glioma samples ranging from 0.1 to 0.87, which have shown significant difference between cisplatin‐sensitive samples and cisplatin‐resistant samples (p < 0.05). The relative expression levels of ERCC1 mRNA in 32 glioma samples were also variable from 0.01 to 5.71. No detectable or low expression of ERCC1 protein was shown in 7 glioma samples. ERCC1 promoter methylation was inversely correlated to mRNA expression (r = −0.903 p = 0.001) as well as protein expression (r = −0.884 p = 0.001). Moreover, ERCC1 mRNA expression was significantly associated with protein levels (r = 0.840 p = 0.001). In summary, the aberrant CpG island methylation in ERCC1 promoter region exists in human glioma cell lines as well as clinical glioma samples. ERCC1 DNA methylation could regulate the expression of downstream mRNA and protein, and was associated with cisplatin chemosensitivity.

Background CD44 is a molecular marker associated with cancer stem cell populations and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting glioma cells high in CD44. Methods Glioma-initiating cell lines were derived from fresh surgical glioblastoma samples. Expression of tissue transglutaminase 2 (TGM2) was attenuated through lentivirus-mediated short hairpin RNA knockdown. MTT assay [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to evaluate the growth inhibition induced by TGM2 inhibitor. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling was used to evaluate cell apoptosis following TGM2 inhibition. CD44⁺ glioma stem cells were sorted by flow cytometry. A nude mice orthotopic xenograft model was used to evaluate the in vivo effect of TGM2 inhibitor. Results TGM2 was highly expressed in CD44-high glioblastoma tissues and tumor-derived glioma-initiating cell lines. TGM2 knockdown impaired cell proliferation and induced apoptosis in CD44-high glioma-initiating cell lines. Further studies indicated that expression of inhibitor of DNA binding 1 protein (ID1) is regulated by TGM2 and might be an important mediator for TGM2-regulated cell proliferation in CD44-high glioma-initiating cell lines. TGM2 inhibitor reduces ID1 expression, suppresses cell proliferation, and induces apoptosis in CD44-high glioma-initiating cell lines. Furthermore, TGM2 is highly expressed in CD44⁺ glioma stem cells, while pharmacological inhibition of TGM2 activity preferentially eliminates CD44⁺ glioma stem cells. Consistently, TGM2 inhibitor treatment reduced ID1 expression and induced apoptosis in our orthotopic mice xenograft model, which can be translated into prolonged median survival in tumor-bearing mice. Conclusions TGM2 regulates ID1 expression in glioma-initiating cell lines high in CD44. Targeting TGM2 could be an effective strategy to treat gliomas with high CD44 expression.