| Glioblastoma multiforme(GBM) is the most common primary brain tumor in adult.Despite Stupp management with surgery, radiation, and chemotherapy,the5-yearsurvival rate is only9.8%[1]. Currently, Temozolomide (TMZ) is the first-linechemotherapeutic agents, making the median survival of newly diagnosed GBMincreased from12.1months to14.6months. However, due to intrinsic and acquiredresistance in GBM, the efficacy of TMZ is limited. Glioma Initiating Cells(GIC) is asmall subpopulation of tumor bulk that possess self-renewal potential anddifferentiation, and are responsible for maintaining the tumor clone.GIC areconsidered to have the capacity to repopulate tumors and mediate radio-andchemoresistance driving malignant progression[3]. Therefore,It is necessary todevelop novel strategies to enhance the efficacy of TMZ. Currently, there are manydrugs research of chemotherapy, which has been found that Chinese traditionalmedicine triptolide (TPL) has anticancer effects, including gliomas [4,5], breastcancer, stomach cancer, bladder cancer [6]. And studies have found that TPL canincrease cytotoxic effect of various chemotherapeutic agents[7]. This study is toinvestigate whether triptolide sensitize temozolomide killing glioma cells andglioma-initiating cells, and to explore its mechanism.Part1: Synergistic Cytotoxic Effect of Triptolide Combined with Temozolomideon Glioma CellsObjective: we aimed to study the interaction between TPL and TMZ in glioma cellsand explore the potential mechanism. Methods: Growth inhibition effect of TMZ and TPL against glioma cells wasmeasured using CCK-8assay. The combined effects between TPL and TMZ wascalculated from Chou-Talalay method. The change of apoptosis and cell cycle andits related protein treated by TPL and/or TMZ in glioma cells was assessed withflow cytometry and western blot.Results: We found that TPL and TMZ both reduced the growth rate of glioma cells ina dose-dependent manner. Triptolide synergistically enhances temozolomidecytotoxicity in glioma cells. Combination index (CI) values of TPL and TMZ are0.76and0.866in U87and SKMG-1respectively, indicating the synergistic effect of thetwo agents. Co-treatment with TPL significantly increased the percentage of apoptoticcells（p<0.05）. Co-treatment synergistically repressed the phoshorylation of IκBα andanti-apoptotic protein XIAP.Conclusions:Triptolide synergistically enhances temozolomide cytotoxicity in gliomacells, which is very likely to be resulted from synergistically enhancing apoptosis bythe augmented repression of NF-κB signaling.Part2: Synergistic Cytotoxic Effect of Triptolide Combined with Temozolomideon Glioma Initiating cellsObjective: we aimed to study the interaction between TPL and TMZ in gliomainitiating cells (GICs) and explore the potential mechanism.Methods: Growth inhibition effect of TMZ and TPL against glioma initiating cellswas measured using CCK-8assay. The combined effects between TPL and TMZ wascalculated from Chou-Talalay method. Self-renewal affected by TPL and TMZ wasevaluated with tumor sphere formation assay. The change of apoptosis treated by TPLand/or TMZ in glioma initiating cells was assessed with flow cytometry and westernblot. Luciferase assay was used to measure NF-κB transcriptional activity.Theexpression of NF-κB downstream genes, the phoshorylation of IκBα and p65and theNF-κB nuclear translocalization were dectected using western blot.Results: We found that TPL and TMZ both reduced the growth rate of glioma initiating cells in a dose-dependent manner,however, GSC-1and GSC-2were resistantto TMZ, with the expected IC50of779.5umol/l and943.5umol/l respectively.Triptolide synergistically enhances temozolomide cytotoxicity in glioma initiatingcells. Triptolide can enhances temozolomide cytotoxicity and reduces the dose ofTMZ. Co-treatment with TPL significantly suppressed the tumor sphere formationand increased the percentage of apoptotic cells. Co-treatment synergistically repressedthe NF-κB transcriptional activity and the expression of NF-κB downstream genes,the phoshorylation of IκBα and p65and the NF-κB nuclear translocalization weresignificantly inhibited.Conclusions: Triptolide synergistically enhances temozolomide cytotoxicity inglioma initiating cells, which is very likely to be resulted from synergisticallyenhancing apoptosis by the augmented repression of NF-κB signaling.Therefore, TPLis a potential chemosensitizer in the treatment of GBM. |
