| Background:As the most common brain tumor in the central nervous system, glioma accounts for35-50%of intracranial tumors in adults. In addition, malignant glioma accounts for about60%of glioma. Because of the biological characteristic of infiltrative growth, glioblastoma is the most aggressive glioma which difficult to eradicate by surgical therapy separated. With the high rate of recurrence, the averaged survival time of patients is only14months. Currently, surgical intervention with adjuvant radiotherapy, chemotherapy and biotherapy are still the major treatment strategies for glioma. However, only a small part of patients could benefit from dose-intensified temozolomide treatment with addition of lomustine, at the cost of increased toxicity, besides, chemotherapy does not significantly prolong the survival of patients with brain glioma because of multidrug resistance (MDR). Therefore, there is urgent need to look for new therapeutic strategies and explore its further mechanisms to improve the therapy for glioma. Glycyrrhizic acid (GA) is a natural and major pentacyclic triterpenoid glycoside of licorice roots extracts. It is commonly used for prevention and treatment of chronic viral hepatitis. Researches have shown that GA exhibits antiulcerative, expectorant, antiviral, antiinflammatory, antidiabetic, anticancer, neuroprotective and immune-enhancing properties. GA could also induce apoptosis via down-regulation of NF-κB (p65) expression in several cancer cell lines such as human hepatoma, promyelotic leukemia, stomach cancer and prostate cancer. GA can also improve anticancer activity of a variety of anticancer drugs, such as cyclophosphamide, vincristine, camptothecin and reduce the toxic side effects of chemotherapeutic drugs, reduce the side effects of chemotherapy. However, the effects of GA on glioblastoma cells had not been reported. In vitro and in vivo studies show that NF-κB is a crucial multi-dominant transcription factor participating in the pathologic process of several human diseases and controls multiple genes involved in tumorigenesis of diffuse glionias and growth promotion of high-grade gliomas. Growing evidence has demonstrated that NF-κB is constitutively activated in carcinogenesis, including glioma. The activation of NF-κB can be detected in all periods of astrocytoma and High level astrocytoma. NF-κB activation levels in recurrent glioma was significantly higher than primary glioma, suggesting that NF-κB may be associated with glioma recurrence. Moreover, activation of NF-κB, which predominantly occurs via the release of the p50/p65heterodimer from the inhibitor of κB and the following translocation of p50/p65heterodimer into the nucleus, leads to the transcriptionalactivation of a variety of genes, including genes encoding cytokines and adhesion molecules, and other genes involved in tumorigenesis and progress. The promotor of Fas-L, IFN-β,IL-8, IP-10and enhancer of HIV are the most common DNA-binding sites of NF-κB. We noted that NF-κB was a crucial transcription factor associated with the occurrence, development, invasion, metastasis, recurrence, apoptosis, immune response of glioma. Besides, GA induces apoptosis in prostate cancer cell lines by down-regulation of p65expression. Hence, we tried to study the effects of GA on p65, the active subunit of NF-κB, in the nucleus of U251cells.Objective:To observe effects of Glycyrrhizic acid on proliferation and apoptosis in human glioma U251cells; explore the possible mechanism via measuring the expression of p65in the nucleus, the active subunit of nuclear factor kappa B(NF-κB), which provides theoretical basis and experimental evidence for therapy of glioma.Methods:1Cell culture:human glioma U251cells were cultured in DMEM medium containing10%fetal bovine serum (FBS) in37℃,5%CO2incubator after cell recovery. The culture medium was changed every2days and the cells were passaged every3-4days. We took U251cells in logarithmic growth phase for experiments.2Detection of cell viability by CCK-8method:U251cells in logarithmic growth phase were digested and suspended, then the cells were counted, followed by inculation into96-well plates, with each well added100μl culture medium containing3×103cells. After cells adherenting, the cell culture solution was replaced with the control and varying concentrations of glycyrrhizic acid solution. The glycyrrhizic acid concentration of each experimental groups were1,2,4mM, each drug concentration groups and control group contained3wells, and cultured them in37℃,5%CO2incubator24,48,72h respectively, each well was incubated with10μl CCK-8solution at37℃for2h. Thereafter, the optical density (OD) for each well was measured at450run using a micoplate reader. Each experiment was repeated three times and the mean value was considered. The cell survival rate was calculated according to the following equation:cell survival rate=(OD experiment-OD blank)/(OD control-OD blank)×100%, draw growth curves and make statistical analysis.3Plate colony-forming assay:Colony-forming is a common method of measuring cell proliferation. Individual cells passage for more than6times in vitro form one clone. The size of a clone is about0.3-1.0mm. Cells in logarithmic growth phase were digested into single cell suspension with trypsin-EDTA solution and then seeded in each25cm2plastic cell culture dishes. They were cultured in growth medium supplemented with GA (0,1,2,4mM) for10days, discarded the medium and then washed2times with PBS, followed by methanol fixing for30min, staining with crystal violet, all purple spots were counted under a magnification of100times.4Assays of cell apoptosis:According to the resultes of cell viability, we selected48h as the particular time point for apoptosis assays.4.1Cell morphology was observed by the inverted phase contrast microscope, and nuclear morphology was analyzed by fluorescence microscope, after staining with Hoechst33258in U251cells:Control and GA-treated U251cells were washed with PBS thrice and then stained with10μl Hoechst33258nuclear dye for10min at room temperature in the dark. After the cells were washed again with PBS thrice, the nuclear morphology was observed by fluorescence microscope with a340nm excitation filter and captured.4.2Annexin V-FITC/PI apoptosis detection kit was used to assay the apoptosis of U251cells:the cells were centrifuged, washed with cold PBS and resuspended in100μl binding buffer. Fluoresce isothiocyanate conjugated Annexin V-FITC (2μl) and propidium iodide (2μl) were added to each sample, and the mixture was incubated at4℃in the dark for15min. The cells were immediately subjected to flow cytometry analysis. The percentages of early and late apoptotic cells in each group were determined.5Western blot analysis:U251cells were treated with various concentrations (0,1,2,4mM) of GA for48h, and proteins from the nucleus were prepared. Equivalent amounts of proteins (50lg per lane) were loaded on gels. Protein samples were separated on8%SDS-PA gels, transferred on a polyvinylidene fluoride membrane and blocked with5%skim milk. Blocked membranes were probed with rabbit anti-human-p65overnight at4℃, washed several times and incubated for1h with anti-rabbit peroxidase-conjugated antibody. The blotted protein bands were visualized by enhanced chemiluminescence and exposed to X-ray film. The optical density of the resulting bands was determined by UN-SCAN-IT, with normalization of densitometry measures to histone H3.6Immun ofluore scence analysis:the U251cells seeded onto a96-well plate were treated by2 mM GA for48h. Cells were fixed in4%paraformaldehyde for15min, and subsequently blocked with10%goat serum in PBS for1h. The cells were incubated with anti-p65antibodies at4℃overnight. The next day, cells were washed three times in PBS for20min and then incubated with Alexa Fluor488conjugated goat anti-rabbit (1:500). Nuclei were counterstained with DAPI and imaged on a fluorescence microscope.7Statistical analysis:Statistical analysis was performed by SPSS16.0statistical software. Data with error bars represent triplicate experiments (mean±SD) unless otherwise noted. P values were determined using one-way ANOVA. A value of P<0.05indicated statistical significance.Results:1Inhibition of proliferation and colony formation by glycyrrhizic acid on U251cells:1.1GA could suppress proliferation of U251cells in a time-and concentration-dependent manner:the IC50of GA on U251cells at24,48and72h were3.67,1.37and1.09mM;1.2After cells were seeded onto25cm2plastic cell culture dishes and cultured for10days, the rate of colony formation in GA treatment groups were:91.6±4.4,81.2±5.0,45.5±5.1and30.7±6.5%respectively, corresponding to the concentration of GA at0,1,2and4mM. Colony counting indicated that the inhibition rate of colony formation presented a concentration-dependent trend.2Glycyrrhizic acid induces apoptosis in U251cells: apoptosis induction by GA was observed in U251cells via Hoechst33258staining and flow cytometry analysis.2.1we observed nuclear morphological and cell morphology changes using fluorescence microscope and the inverted phase microscope, respectively. U251cells in control group were irregular fusiform and had much synapasis, good diopter, abundant cytoplasm and clear adge, and bonded to the culture plate closely. Nuclei in the control group were round and homogeneously stained, while these became round, condensed and fragmented after treatment with2mM GA for48h and displayed marked granular apoptotic bodies. According to this feature of apoptosis, we assumed that GA induced apoptosis of U251cells;2.2The degree of apoptosis was measured by flow cytometry. The percentage of apoptotic cells in GA treatment groups were:3.30±0.98,14.97±2.67,27.53±5.51and55.90±5.1%, respectively, corresponding to the concentration of GA at0,1,2and4mM. The apoptosis rate of U251cells increased when treated with an increased concentration of GA.3Expression and distribution of p65in U251cells after GA treatment:p65protein was expressed at a very high level in the control group (2.83±0.38), while in GA-treated groups, the protein levels of p65significantly decreased (1.91±0.31,1.43±0.28,0.94±0.22). There was a statistically significant difference between the control group and each GA group (1mM, P<0.05;2mM, P<0.01;4mM, P<0.01). In addition, immunofluorescence staining with an anti-p65antibody confirmed the robust expression of p65located in the nucleus in the control group, while p65staining was weak after2mM GA treatment for48h.Conclusions:Our present study suggests that GA addition confers anticancer efficacy by inhibiting the proliferation and inducing apoptosis in human glioblastoma U251cells. These findings provide an important implication for the use of GA as an antiglioblastoma drug in clinical trials. Moreover, the suppression of NF-κB by GA may provide a potential target for the prevention and treatment of many NF-KB-associated disorders. |
PDF Full Text Request