| Background and objective：Glioma, accounting for40%to50%brain tumors, is the most commonmalignancy in central nervous system and hard to cure, with characteristics of highrecurrence rate, high mortality and low cure rate in clinical. The prognosis is poor,with a3~5years of average survival time in low-grade gliomas (WHO I-II level) and1to2years average survival time in high-grade gliomas (WHO III-IV-class), and theoverall5year survival rate after operation is less than10%. The main treatmentoptions currently in glioma is surgery and radio-chemotherapy, and the chemotherapydrugs commonly used are nitrosourea alkylating agents, vincristine, temozolomide,etc. Although chemotherapy drugs demonstrate some effects, the survey shows thatthe side effects is severe, and often lose efficacy when the glioblastoma relapse again.So looking for more efficient and less toxic drugs is a direction to explore bestmethod for the treatment of glioma in current. Chinese traditional medicine is a brightpearl of medicine, and it has played an important role in the health guard of peoplefor thousands of years. Chinese licorice root and stems can be used as medicine, withthe medical effect of spleen Qi, relieve pain, cough expectorant, detoxification, andreconcile the various drugs and other effects. Isoliquiritigenin is one of itscomponents, with a class isoflavones as yellow needles crystallization. Studies haveshown that isoliquiritigenin has certain antitumor efficacy, such as prostate cancer,cervical cancer, and also has tumor chemopreventive effects, reducing the side effectsof chemotherapy to the liver and kidney. This study is designed to investigate the effects of Chinese medicine isoliquiritigenin on suppressing glioma cell proliferationand invasion and metastasis, and to explore the combined effects with chemotherapydrug cisplatin in glioma, which may provide pharmacology basis for isoliquiritigeninin treatment of gliomas.Method：1．In vitro experimental study of isoliquiritigenin in inhibition of glioma cellproliferation, apoptosis and invasion: MTT assay, flow cytometry, Transwell assaywere used to investigate the antitumor effects with different concentrationsisoliquiritigenin in U87and C6glioma cells; Western blot method was used to detectthe expression of caspase-3, MMP-2, MMP-9, p21, p27, Akt, p-AKT protein.2．Experimental study of isoliquiritigenin combined with cisplatin in glioma:MTT assay, flow cytometry were used to assay the glioma cell proliferation, cellcycle and apoptosis; Western blot method was used for detecting changes incaspase-3, bcl-2, bax and other key apoptotic molecules; Glioma C6xenograft andorthotopic model were used to evaluate the antitumor effect of isoliquiritigenin incombination with cisplatin; Immunohistochemical analysis was used to detect theexpression of caspase-3and ki-67.Results：1．MTT test results showed that the inhibition rate of isoliquiritigenin on gliomaU87and C6cells was increased with the increasing of concentration (0,5,10,20,40,80μmol/L). The IC50of isoliquiritigenin for glioma U87cells in24h,48h and72hwere115.0,38.8and22.1μmol/L respectively, while the IC50of isoliquiritigenin forC6cells in24h,48h and72h were108.5,37.4and24.3μmol/L respectively. Flowcytometry analysis showed that isoliquiritigenin at a concentration of greater than60μmol/L caused a significant S and G2/M phase arrest. Annexin V/PI doublestaining showed that apoptosis of glioma cells could be induced by isoliquiritigeninwith early and late apoptosis and necrosis. Adding of broad-spectrum caspaseinhibitor Z-VAD-FMK did not influence the effect of cell necrosis induced by isoliquiritigenin. Transwell results showed that ISL of different concentrations (0μmol/L,20μmol/L,40μmol/L and60μmol/L) acting on the U87glioma cellscaused a inhibition ratios of U87cell invasion of0,17.2%,33.8%and57.3%respectively. Western blot analysis showed that isoliquiritigenin of differentconcentrations (0μmol/L,20μmol/L,40μmol/L and60μmol/L) acting on U87glioma cells reduced the MMP-2/9, p-Akt expression levels and increased theexpression of p21, p27and caspase-3and other proteins.2．MTT test results showed that isoliquiritigenin enhanced the anti-glioma effectof cisplatin, and the joint index of them was better when cisplatin was6.25μmol/Land12.5μmol/L and isoliquiritigenin was20μmol/L. The results of flow cytometryshowed that the combination of cisplatin (6.25μmol/L) with isoliquiritigenin (20μmol/L) increased the S phase cycle arrest significantly, and the apoptotic ratio wasincreased from4.32±1.07%and2.25±0.44%to13.2±1.52%. Western blot analysisshowed that isoliquiritigenin combined with cisplatin upregulated the expression ofcaspase-3and bax and downregulated bcl-2expression levels. In vivo experimentsshowed that isoliquiritigenin enhanced the anti-tumor efficacy of cisplatin. Theinhibition efficiency of glioma of isoliquiritigenin at500μg/kg and that of cisplatinat concentration of1mg/kg were31.0%and48.6%respectively, while thecombination of them increased anti-tumor efficacy to66.9%. In orthotopic gliomamodel, combination of isoliquiritigenin with cisplatin was able to prolong survival inrats significantly (compared with the control group, P <0.05). Immunohistochemicalanalysis showed that combination of isoliquiritigenin with cisplatin increased theexpression of Caspase-3(compared with the control group, P <0.01), while itdecreased ki-67antigen expression (compared with the control group, P <0.01).Conclusion：1．Isoliquiritigenin at10~80μM concentration is able to inhibit the proliferationof glioma U87and C6cells in dose-dependent manner and induce G2/M phase and Sphase arrest and apoptosis, which might caused by down-regulation of MMP-2/9, Aktphosphorylation levels and other factors; 2．Isoliquiritigenin enhance the anti-tumor effect of cisplatin without increasingof system toxicity, which may be associated with influencing the expressing ofcaspase-3, bcl-2, bax, and Ki-67protein levels, etc. |
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