Temozolomide (TMZ) can cross the blood-brain barrier (BBB) and deliver methyl groups to the purine (guanine) bases of DNA, leading to mispairing during DNA replication and subsequent cell death. However, increased expression of the repair enzyme methyl guanine methyltransferase (MGMT), which removes methyl groups from purine bases, counteracts methylation by TMZ. We evaluated the anticancer potential of thymoquinone (TQ), a hydrophobic flavonoid that inhibits resistance and induces apoptosis in various cancer cells, both in vitro and in vivo. In vitro experiments showed that compared with the Hs683 and M059J cell lines, U251 cells were more sensitive to TMZ. Compared to U251 cells, U251R cells, a TMZ drug-resistant strain established in this study, are characterized by increased expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and MGMT. TQ treatments induced apoptosis in all cell lines. The p38 mitogen-activated protein kinase signal pathway was mainly activated in U251 and U251R cells; however, p-ERK and MGMT upregulation could not suppress TQ effects. Furthermore, si-p38 pretreatment of U251R cells in TQ treatments inhibited cell apoptosis. We speculate that TQ contributed to the phosphorylation and activation of p38, but not of ERK-induced apoptosis (irrespective of TMZ resistance). In vivo, U251R-derived tumors subcutaneously inoculated in nude mice exhibited significant tumor volume reduction after TQ or TQ + TMZ cotreatments. High-performance liquid chromatography assay confirmed the presence of TQ in murine brain tissues. Our findings demonstrate that TQ can effectively cross the BBB and function alone or in combination with TMZ to treat glioblastoma.

Background Neutrophils form the first line of innate host defense against invading microorganisms. We previously showed that F0F1 ATP synthase (F-ATPase), which is widely known as mitochondrial respiratory chain complex V, is expressed in the plasma membrane of human neutrophils and is involved in regulating cell migration. Whether F-ATPase performs cellular functions through other pathways remains unknown. Methods Blue native polyacrylamide gel electrophoresis followed by nano-ESI-LC MS/MS identification and bioinformatic analysis were used to identify protein complexes containing F-ATPase. Then, the identified protein complexes containing F-ATPase were verified by immunoblotting, immunofluorescence colocalization, immunoprecipitation, real-time RT-PCR and agarose gel electrophoresis. Immunoblotting, flow cytometry and a LPS-induced mouse lung injury model were used to assess the effects of the F-ATPase-containing protein complex in vitro and in vivo. Results We found that the voltage-gated calcium channel (VGCC) α2δ-1 subunit is a binding partner of cell surface F-ATPase in human neutrophils. Further investigation found that the physical connection between the two proteins may exist between the F1 part (α and β subunits) of F-ATPase and the α2 part of VGCC α2δ-1. Real-time RT-PCR and PCR analyses showed that Cav2.3 (R-type) is the primary type of VGCC expressed in human neutrophils. Research on the F-ATPase/Cav2.3 functional complex indicated that it can regulate extracellular Ca2+ influx, thereby modulating ERK1/2 phosphorylation and reactive oxygen species production, which are typical features of neutrophil activation. In addition, the inhibition of F-ATPase can reduce neutrophil accumulation in the lungs of mice that were intratracheally instilled with lipopolysaccharide, suggesting that the inhibition of F-ATPase may prevent neutrophilic inflammation-induced tissue damage. Conclusions In this study, we identified a mechanism by which neutrophil activity is modulated, with simultaneous regulation of neutrophil-mediated pulmonary damage. These results show that surface F-ATPase of neutrophils is a potential innate immune therapeutic target. Graphical abstract