Compared to standard therapy, the antitumor effects of rendezvous therapy were more effective in GBM patients without increasing the toxicities.
To investigate the effect of valproic acid (VPA) on the susceptibility of glioma stem cells to temozolomide (TMZ) and nimustine (ACNU), the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and its expression of MGMT were examined. A total of 3 glioma cell populations were isolated from human glioma tissues, and immunocytochemistry was used to detect the expression of MGMT. VPA inhibition on the growth of the 3 glioma cell populations exposed to various concentrations of TMZ and ACNU was evaluated. Flow cytometry was applied to detect the apoptosis of glioma cells, and a methylation-specific polymerase chain reaction was used to identify methylation of MGMT promoter. Immunocytochemistry results indicated that MGMT was negatively expressed in the G1 population, but positively expressed in the G2 and G3 populations. Cell growth inhibition assays demonstrated that the survival rate in the VPA + TMZ or ACNU groups was decreased compared with that of the TMZ or ACNU alone groups (P<0.05). As for the apoptotic rate, those in the VPA alone group were increased compared with the control group (P<0.05), and the rates in the VPA + TMZ or ACNU groups were increased compared with TMZ or ACNU alone groups (P<0.05). The expression of MGMT remained negative in the G1 population following treatment with VPA, but MGMT expression became negative in the 2 MGMT-positive cell populations (G2 and G3) following VPA treatment. The MGMT promoter in the G1 population was partially methylated in the control group, but was fully methylated following VPA treatment, while the promoters of G2, G3 were unmethylated in the control group and became partially methylated in the VPA treatment group. Taken together, TMZ and ACNU may suppress the growth of glioma stem cells in vitro in a dose-dependent manner. VPA may enhance the inhibitory effects of various concentrations of TMZ and ACNU on the growth of MGMT-negative/positive cells, particularly on MGMT-positive cell populations. VPA itself may induce the apoptosis of glioma cells, and VPA combined with TMZ or ACNU may enhance TMZ/ACNU-induced apoptosis of glioma stem cells. Furthermore, VPA may also promote the methylation of the MGMT promoter to silence MGMT expression in glioma cells, which may be an important mechanism through which VPA enhances the efficacy of TMZ and ACNU in targeting glioma stem cells.
In the present study, to delve into the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism, six cases of high-grade glioma cells from patient’s tumor tissues were cultured. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed to detect cell proliferation and toxicity. Flow cytometry was performed to ascertain cell cycle and apoptosis rate. To detect the expressions of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein, respectively, specific PCR and immunofluorescence were performed. According to the results of MTT assay, compared with the results of control group, GM-CSF group exhibited enhanced cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the combination group [(67.67 ± 1.16), (68.13 ± 1.06), (68.42 ± 1.73)] had noticeably lower cell viability than the corresponding TMZ group [(90.00 ± 1.73), (82.33 ± 1.53), (82.67 ± 2.11)] (P < 0.01). Nevertheless, the two groups showed no significant difference in another three cases (MGMT gene unmethylated) (P > 0.05). In combination group, the apoptosis rate of the MGMT methylation cells was higher than that in the corresponding TMZ group (P < 0.01), which is consistent with MTT assay results. In all six cases of primary glioma cells, the fraction of cells in G1 phase of GM-CSF-treated group was noticeably down-regulated and was up-regulated in S phase (P < 0.01). GM-CSF could induce high-grade glioma cells to rapidly enter the cell cycle, thereby enhancing the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation. However, this effect is not ideal on glioma cells with MGMT unmethylation.
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