Liquiritigenin Enhances The Sensibility Of The Apoptosis Promoting Effect Of Temozolomide On Glioblastoma Cells

Background:Temozolomide(TMZ) is an alkylating agent used as a first-line treatment for glioblastoma multiforme. It could efficiently penetrate the blood brain barrier, and quickly converted to antitumor active substances with strong cytotoxic effects. However, the therapeutic efficacy of TMZ remains ineffective as the inherited or acquired drug resistance is frequently observed.Therefore, how to solve the problem about TMZ resistance, restore the sensitivity of tumor cells to drug treatments, it becomes the new hot spot of neurosurgery clinicians and researchers. Estrogen receptor β(ERβ) is a new subtype of estrogen receptor, it is a ligand regulated transcription factor type.It is a multifunctional protein associated with the development and differentiation of nerve cells, with tumor suppressor gene activity. ERβ has emerged as a tumor suppressor and a key regulator of signal transduction in glioma cells. However, little is known about the role of ERβ in regulating chemotherapeutic response to TMZ. Therefore, we use ERβ agonists Liquiritigenin(Liq) to enhanced the sensitivity of TMZ to glioma cells, and study the mechanism of Liq sensitized TMZ.Methods:This study include two parts in vivo and in vitro. In vitro study, we selected three human glioma cell lines: U138 is low sensitivity to TMZ, U251 is high sensitivity to TMZ, T98 G is resistant to TMZ, Effects of proliferation,apoptosis and cell cycle of glioma cells were detected using MTT assay and flow cytometry after treated with Liq alone or combination with TMZ;expressed levels of protein and phosphorylation of apapoptotic factors,signaling molecular of ERβ, MGMT, Caspase-3, Akt, p-AKT, P70S6 K,p-P70S6 K, and so on were examined using western blot; RNA interference was used to silence the expression of ERβ, and the mechanism of Liq effected on PI3K/AKT/m TOR signaling pathway was clearly defined; combination of activitors and inhibitor of PI3K/AKT/m TOR pathway to further explore the role and the molecular mechanisms of Liq regulated the pathway and promoted TMZ sensitivity. In vivo study, glioma model in nude mice was taken as the research object, U138 cells(106/100μl) was injected into the left abdominal subcutaneous inoculation in nude mice for 7 days, then they were divided into four groups: control group(DMSO), TMZ group(50 mg/kg/d),Liq group(30 mg/kg/d), and combination group(TMZ 50 mg/kg/d+Liq 30mg/kg/d), the growth of tumor in nude mice was observed, Liq effected the expression of ERβ and the synergistic response of TMZ were studied in vivo.Results:In vitro: Glioma U138 cells were treated with different concentrations of Liq(10, 20, 40, 80, 160 and 320μM) for 72 h, Liq significantly inhibited the cell viability in a dose-dependent manner, and the inhibition increased with concentration increased. As 80 μM Liq was sufficient to activate ERβ without causing severe cell death in U138 cells, this concentration of Liq was used in all subsequent assays. After combined treatment of different concentration of TMZ,Liq can enhance the inhibition of U138 cells to TMZ. Cell viability was only50.36% after 100 μM TMZ and 80μM Liq combined treatment for 72 h, it significantly lower than treatment of TMZ alone(90.33%, P < 0.001), it proved that Liq can effectively enhance the sensitivity of tumor cells to TMZ. Cell cycle analysis showed that Liq can further enhance S-phase block by TMZ induced(64.63% vs 40.43%, P < 0.001), and effective inhibit cells into the proliferated phase, when compared with TMZ alone. Annexin V/PI analysis showed that the combination of Liq and TMZ can obviously increase the apoptotic rates(27.5% vs 18.6%,P<0.001), and also increase the formation of cleavage of Caspase-3. The synergistic effect of Liq and TMZ was also verified in the other two types of glioma U251 and T98 G cells. Western blot results showed that Liq can activate ERβ, and decrease the levels of phosphorylation of Akt and P70S6 K, and then inhibit the activation of PI3 K / Akt / m TOR pathway,but there is no direct impact on MGMT. Liq lost the ability of regulation to PI3K/AKT/m TOR pathway after silencing ERβ expression by RNA interference,it indicated that Liq is regulated PI3K/AKT/m TOR pathway by the activation of ERβ. XL765 is the inhibitor of PI3K/AKT/m TOR pathway, it can significantly enhance the sensitivity of cells to TMZ, it suggested that the activation of the PI3K/AKT/m TOR pathway has a protective effect on glioma cells, which antagonize the effect of TMZ. IGF-1 as the activator of PI3K/AKT/m TOR pathway can inhibit TMZ toxicity that it was mediated by Liq, it showed that Liq play a role through the inhibition of PI3K/AKT/m TOR pathway.In vivo: The results showed that the mean tumor volume of control group was1.84±0.13cm3, TMZ group was 0.93±0.11cm3, Liq group was 1.34±0.15cm3, and combination of TMZ and Liq group was 0.36±0.04cm3. This result is consistent with the results of in vitro, the combination of Liq and TMZ can significantly inhibit the growth of tumor, and increase the tumor sensitivity to TMZ, the difference was statistically significant(P < 0.05). At the same time, western blot results showed that Liq can increase the expression of ERβ of tumor cells in vivo, so we thought Liq could activate ERβ and the related signaling pathways to inhibit the growth of tumor, also improve the effects of tumor cell sensitivity to TMZ.Conclusion:1. Liq could inhibit the proliferation and significantly promote the apoptosis of glioma cells(U138), and also could obviously enhance the sensitivity of tumor cells to TMZ;2. Liq inhibited the activation of PI3K/AKT/m TOR signaling pathway through specifically activated by ERβ;3. The enhanced sensitivity of Liq to TMZ is dependent on inhibion of PI3K/AKT/m TOR signaling pathway;4.The studies in vivo showed that Liq could significantly increase the sensitivity of tumor cells to TMZ when Liq and TMZ combination in bearing mice.These results suggest that Liq can promote the effect of TMZ by activating ERβ to inhibit PI3K/AKT/m TOR in the treatment of glioma. At the same time, this study also provides evidence and clues for other ERβ agonists as an effective chemotherapeutic agent for the treatment of glioma.