Experimental Studies On The Therapeutic Effects Of Quetiapine,an Antipsychotic Drug On Glioma

Malignant glioma cells are not sensitive to radiotherapy and chemotherapy,resulting in low clinical cure rate,high recurrence rate and less than 5% of five-year-survival rate of patients.Therefore,it is very important to find an effective and innovative biological therapy.In recent years,the introduction of the concept glioma stem-like cells(GSLCs)has provided an important target for the treatment.Found in glioma,though only of a very small number,these cells are similar with the neuronal stem cells(NSCs),which are self-renewal,persistent growth.These cells can also express NSCs markers like Nestin and CD133.Besides,they are highly resistant to radiotherapy and chemotherapy.The GSLCs can differentiate into cells which phenotypes are similar with neurons,astrocytes or oligodendrocytes.The differentiated GSLCs have a lower capability to form a tumor and are much more sensitive to chemotherapy and radiotherapy.Therefore,finding a drug to promote the differentiation of the GSLCs may provide a novel way for clinical application against the glioma.Epidemiological research revealed an interesting phenomenon: the incidence of cancer in schizophrenia patients was obviously lower compared with normal people.Researchers found that many antipsychotic drugs,such as chlorpromazine,olanzapine and risperidone,can significantly inhibit the proliferation of the glioblastoma cell line IMR32 but had little effect on normal cells.Quetiapine(QUE)is the fourth atypical antipsychotic developed after clozapine,risperidone and olanzapine.Previous study of our group firstly found that QUE could significantly increase the ratio of oligodendrocytes differentiated from the NSC cultures and promote the maturation of oligodendrocyte progenitor cells(OPCs)and myelination.Considering that GSLCs have similar biological properties with the NSCs and OPCs,we raised up the hypothesis that QUE may affect the proliferation and differentiation of the GSLCs and inhibit the gliomagenesis and its development.Based on this hypothesis,the research first tested the effect of QUE on glioma by filtering GSLCs in vitro and building up a tumor model,QUE application was deemed as the treatment and in some group first-line drug against glioma Temozolomid(TMZ)was also used.Next,by setting up in vitro GSLCs model and inducing proliferation or differentiation by QUE,the research deeply surveyed the molecular mechanism behind QUE's inhibition of the proliferation and the positive effect on differentiation on the GSLCs.The research is composed of three parts:Part 1.Effects of QUE on GSLC-originated gliomagenesisGSLCs can be purified from the glioblastoma cell line GL261 by using serum free conditional medium.Then they were used to set up the xenograft model in nude mice and the orthotopic transplantation model in C57 mice.Grouping was based on the application of the drugs: namely QUE,TMZ,QUE&TMZ and the control group.Techniques for detecting the effects of various treatments on the incidence and growth characteristics of glioma in the experiments include bioluminescence imaging,behavioral experiments,HE staining,and immunohistochemistry.After xenograft subcutaneous and vertical gliomagenesis,TMZ was given for 21 days to kill the tumor cells.Then TMZ was withdrawn and the mice were treated with QUE.Bioluminescence imaging was applied to detect the condition of the glioma.Main results are listed as below:1.GSLCs were filtrated from the glioblastoma cell line GL261 cultured in serum-free medium.Immunofluorescence result showed that the GSLCs expressed the stem markers CD133,Sox2,NG2 and Nestin at a high level,indicating that the filtration of GSLCs was successful.2.The GSLCs acquired were used for subcutaneous transplantation and treated respectively by QUE,TMZ and QUE&TMZ.After 7 days a small tumor was formed in control group.After 21 days the average diameter of the tumor reached 1.5cm.While the tumor failed to form in treatment group until 10 days later than the control,the average volume of the tumor was also smaller.Among these groups QUE&TMZ group showed the best effect.Histopathological examination showed that there were less mitotic figures in glioma cells of the treatment group and fewer proliferating cell nuclear antigen(PCNA)positive cells.Compared with the control,there was apparently less Olig2 positive cells but more MBP positive cells.Western blot result showed that in QUE and QUE&TMZ group the expression of MBP was obviously higher,while Vimentin and GFAP expression has been reduced(P <0.05).3.The GSLCs acquired were used for vertical transplantation and treated respectively by QUE,TMZ and QUE&TMZ.The result was similar with that in subcutaneous transplantation: after 7 days tumor were found in mice,while after QUE and TMZ treatment the volume became smaller,with the smallest volume in QUE&TMZ group;survival rate was significantly higher than control.Open Field experiment result showed that the total distance of the treatment group significantly increased(P < 0.05).4.The two models have been repeated and the mice were then treated by TMZ for 21 days,the growth of the tumor was obviously inhibited.After withdrawn of the drug the mice were treated by QUE,the result revealed that in control group the tumor recurred,whose volume was comparable with the tumor volume before treatment.While in QUE group,the growth of the tumor was significantly slower and the tumor volume was also smaller.From these experiments,three conclusions can be determined:(1)GSLCs can be filtrated from serum-free cultures.(2)In tumor models based on GSLCs,both QUE and TMZ can effectively repress tumor grow and lower its malignancy.(3)TMZ mainly inhibit the growth of mature tumor cells while the function of QUE focuses on inhibiting the proliferation and promoting the differentiation of the tumor stem cells.Part2.Effects of QUE on proliferation and differentiation of GSLCsGSLCs were separated in vitro using the same method in Part1.The cells were then cultured in proliferation or differentiation medium and induced by QUE of different concentrations.To verify the effect of QUE on the proliferation and differentiation of GSLCs,the proliferation properties were detected by MTT,RT-PCR,and flow cytometry while the types and ratio of the cells differentiated from GSLCs were detected by immunofluorescence and Western blot.Main results are listed as below:1.Cells cultured in proliferation medium were treated with 0,1,5,10,20,50,100?mol/L QUE.After being treated by 50,100?mol/L QUE,suspension GSLCs' sphere volume apparently became smaller.After being treated by 50?mol/L QUE,MTT detection revealed that the proliferation capability of the cells was significantly reduced;flow cytometry result revealed that S phase GSLCs were significantly reduced;RT-PCR result revealed that in GSLCs Sox2 and Ki67 expression were significantly reduced.2.Cells were cultured in differentiation medium with 1% fetal bovine serum and treated by 25?mol/L QUE.Immunofluorescence result revealed that after QUE treatment in differentiated GSLCs MBP and CC1 positive cell number was significantly increased while GFAP positive cell number was reduced.After being treated by 5,10,25,50 ?mol/L QUE,Western blot result revealed that as with the increasing of QUE concentration,the expression of Sox2 and Olig2 were down-regulated while MBP and Olig1 expression were up-regulated.These results indicate that:(1)in proliferation medium,QUE(50,100 ?mol/L)can effectively repress GSLCs proliferation.(2)In differentiation medium,QUE(25 ?mol/L)can promote the differentiation of GSLCs towards oligodendrocyte-like cells.The effect is relatively concentration-dependent.Part 3.The molecular mechanism of QUE on promotion of GSLC differentiationPrevious research proved that Wnt/?-catenin signaling pathway has an important role inGSLCs' proliferation and differentiation.Part1 and Part2 experiment results also proved that QUE can effectively inhibit tumorigenesis and GSLCs' proliferation,inducing the differentiation towards oligodendrocytes.Is the function of QUE on GSLCs related with Wnt/?-catenin signaling pathway? To further study on this question,experiments were designed for Part3.The effect of QUE on molecules in Wnt/?-catenin signaling pathway was detected for the growth process of the tumor in vivo and for the differentiation of the GSLCs in vitro.Immunohistochemistry and Western blot were applied.Main results are listed as below:1.Immunohistochemistry experiments for the control group in vivo showed that ?-catenin was expressed at a high level in cytoplasm and entered the nucleus,while in QUE group there was a lower expression in cytoplasm.Western blot result showed that in QUE group the expression of p-GSK3?(ser9)has been reduced,and the same case was true for ?-catenin.2.During the differentiation of GSLCs,QUE treatment can lower the expression of p-GSK3?(ser9)and promoting phosphorylation of ?-catenin so as to lower the level of it.This function is time/concentration dependent and can be attenuated by Wnt/catenin signaling pathway agonist QS11 and LiCl.Experimental results in Part3 indicates that QUE can activate GSK-3? and accelerate degradation of ?-catenin,so as to repress Wnt/?-catenin signaling pathway and promote the differentiation process of GSLCs towards oligodendrocyte-like cells.To make a summary,we proved that antipsychotic QUE can inhibit the proliferation of the GSLCs and the growth of the tumor by inhibiting Wnt/?-catenin signaling pathway and promoting the differentiation of GSLCs towards oligodendrocyte-like cells;QUE&TMZ combined application may be a novel strategy against malignant glioma.