| Objective: Malignant glioma is a common central nervous system tumor.The therapeutic efficacy of conventional surgical resection and adjuvant chemotherapy remains unsatisfactory.One important factor contributing to the poor prognosis of patients with glioma is resistance to chemotherapy.However,the specific mechanism responsible for drug resistance is currently unknown.Several hypotheses have been proposed to explain chemotherapy resistance,including the resistance of glioma stem cells(GSCs)to chemotherapy drugs and the abnormal expression of multidrug resistance proteins in gliomas.GSCs are glioma cells with immortal proliferation and multidirectional differentiation potential.P-glycoprotein(P-gp)and adenosine triphosphate binding cassette transporter G2(ABCG2)are multidrug resistance proteins associated with glioma resistance.Inhibiting the proliferation of GSCs and reducing the expression of P-gp and ABCG2 may decrease the drug resistance of gliomas.The role of nuclear factor erythorid-2-related factor 2(Nrf2)and its mediated pathways in the treatment of gliomas has been a research hotspot in recent years.High expression of Nrf2 and associated downstream genes in glioma cells,especially in GSCs,promotes the proliferation of glioma cells and increases the resistance of glioma to chemotherapy drugs.The combination of low frequency and low intensity ultrasound(LFLIU)and curcumin inhibits the proliferation of glioma cells and the expression of multidrug resistance proteins by regulating the PI3K/Akt or Nrf2 signaling pathway.Metformin(MET)has recently been studied for its role in the treatment of glioma.However,whether LFLIU combined with MET can inhibit the proliferation of GSCs and affect the expression of Nrf2,P-gp,and ABCG2 remains to be determined.In this study,based on our previous findings,using in vitro and in vivo experiments,we determined whether LFLIU combined with MET inhibited the proliferation of glioma cells and GSCs,and whether the combined effect reduced Nrf2,P-gp and ABCG2 in GSCs.The effect of Nrf2 on the proliferation of GSCs and the expression of P-gp and ABCG2 was also studied.Methods: 1.U-87 MG-GSCs and U251-GSCs cells were cultured and separated from U-87 MG and U251 using a serum-free culture method.U-87 MG,U251,U-87MG-GSCs,and U251-GSCs cells were treated with LFLIU,MET,and LFLIU+MET.Based on the team's previous research,LFLIU was used at a frequency of 1 MHZ,the ultrasonic irradiation depth was 5 mm,the gain was 5 d B,the intensity was 142.0m W/cm2,the action time was 60 s,and the MET concentration was 10 m M.The CCK8 method was used to detect the effects of LFLIU,MET,and LFLIU combined with MET in U-87 MG,U251,U-87 MG-GSCs,and U251-GSCs.The rate of inhibition of cell proliferation in response to the different treatments was calculated.2.An orthotopic transplantation model of U251-GSCs was established in immunodeficient animals(nude mice).Tumor growth was monitored using nuclear magnetic resonance,and cell morphology and structure were evaluated by HE staining.The localization of Nrf2,P-gp,ABCG2,Ki-67,and VEGF was examined by immunohistochemistry,and the expression of Nrf2,P-gp,ABCG2,Ki-67,and VEGF was analyzed semi-quantitatively.Glioma-bearing nude mice were randomly selected from each intervention group for survival analysis.3.The polymerase chain reaction and western blotting were used to detect the combined effects of LFLIU,MET,and LFLIU+MET on the expression of Nrf2,P-gp,and ABCG2 in U251-GSCs at the gene transcription and protein levels in vitro and in vivo.4.U251-GSCs were transfected with lentivirus to reduce the expression of Nrf2.The CCK8 method was used to detect the effect of U251-GSCs on cell proliferation.The polymerase chain reaction and western blotting were used to detect changes in the expression of P-gp and ABCG2 at the gene transcription and protein levels after the expression of Nrf2 in U251-GSCs was reduced.Results: 1.Glioma spheres containing U-87 MG-GSCs and U251-GSCs were grown in serum-free medium,and the ratio of CD133+ GSCs in glioma spheres was examined by flow cytometry.The average proportion of CD133+ U-87 MG-GSCs in glioma spheres was 0.93%,whereas that of CD133+ U251-GSCs was 0.87%.2.In the U-87 MG group,the cell proliferation inhibition rates in response to treatment with MET,LFLIU,and MET+LFLIU were 22.53 ± 3.18%,19.77 ± 3.03%,and 24.51 ±3.39%,respectively,at 48 h,and 36.27 ± 4.17%,33.24 ± 4.74%,and 48.63 ± 5.15%,respectively,at 72 h.In the U251 group,the cell proliferation inhibition rates in response to MET,LFLIU,and MET+LFLIU were 23.76 ± 3.36%,19.89 ± 3.12%,and 26.58 ±3.51%,respectively,at 48 h after the treatment,and 35.91 ± 4.62%,34.26 ± 4.29%,and50.69 ± 5.82%,respectively,at 72 h after treatment.In the U-87 MG-GSCs group,the cell proliferation inhibition rates after MET,LFLIU,and MET+LFLIU treatment were27.11 ± 3.59%,28.98 ± 3.78%,and 31.38 ± 4.08%,respectively,at 48 h,and 38.88 ±4.37%,35.61 ± 4.47%,and 55.86 ± 6.09%,respectively,at 72 h.In the U251-GSCs group,the cell proliferation inhibition rates of MET,LFLIU,and MET+LFLIU were30.78 ± 3.81%,29.51 ± 3.24%,35.46 ± 4.17%,respectively,after 48 h of treatment and45.39 ± 4.41%,39.78 ± 4.83%,and 68.16 ± 9.15%,respectively at 72 h.At 48 h after the different treatments,the cell proliferation levels did not differ significantly between the groups.At 72 h,the combined application of MET and LFLIU had a significant inhibitory effect on cell proliferation compared with each treatment alone,and the difference was statistically significant(P < 0.05).The inhibitory effect of MET and LFLIU on the proliferation of U251-GSCs in the combined group was more significant than that of U-87 MG,U251,and U-87 MG-GSCs(P < 0.05).3.Treatment with LFLIU,MET,and LFLIU+MET reduced the growth rate of intracranial tumors in tumor-bearing nude mice.The combination of LFLIU+MET was the most effective(P < 0.05).The survival time of tumor-bearing nude mice was longer in the group treated with LFLIU+MET than in the control or single treatment groups(LFLIU group,MET group)(P < 0.05).4.Nrf2,P-gp,and ABCG2 were highly expressed in the control group.Compared with the control group and the single treatment groups(LFLIU group or MET group),the expression of Nrf2,P-gp,and ABCG2 was lower in U251-GSCs at the gene and protein levels in response to combination treatment with LFLIU+MET in vitro and in vivo(P <0.05).5.In the Scrambled group,there was no obvious inhibitory effect on the cell proliferation of U251-GSCs.In the U251-GSCs transfection(U251-GSCs-Nrf2-down)group,there was a significant inhibitory effect on the proliferation of U251-GSCs,and there was a statistically significant difference compared with the Scrambled group(P<0.05).The expression of Nrf2,P-gp,and ABCG2 was significantly lower at the gene and protein levels in the U251-GSCs-Nrf2-down group than in the control group and the Scrambled group(P < 0.05).The expression of Nrf2,P-gp,and ABCG2 did not differ significantly between the Scrambled group and the control group.Conclusions: 1.Combination treatment with LFLIU+MET had a stronger inhibitory effect on the proliferation of glioma cells and GSCs than LFLIU and MET alone.LFLIU+MET can inhibit the growth of intracranial tumors in tumor-bearing nude mice and prolong their survival time.2.LFLIU+MET can inhibit the changes of Nrf2,P-gp,and ABCG2 in U251-GSCs at the gene transcription level and affect the expression of P-gp and ABCG2 at the protein level both in vivo and in vitro.Compared with LFLIU and MET alone,LFLIU+MET may have an important effect on reversing the multidrug resistance of gliomas.3.Combination treatment with LFLIU and/or MET inhibits the proliferation of glioma cells and GSCs,slows down the growth of intracranial tumors in tumor-bearing nude mice,and prolongs their survival time.P-gp and ABCG2 expression at the gene transcription and protein levels may be related to Nrf2 and signaling pathway regulation.4.The relative safety of MET and the selectivity of the mode of action of LFLIU indicate that they may be a promising adjuvant therapy for glioma. |
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