Tumor suppressive effect of low-frequency repetitive transcranial magnetic stimulation on glioblastoma progression

Abstract

Repetitive transcranial magnetic stimulation (rTMS) is used as a non-invasive treatment for various diseases, and its potential application in cancer treatment has been proposed by researchers. However, the precise mechanisms and effects of rTMS on many types of cancer, including glioblastoma (GBM), remain largely unknown. This study aimed to investigate the effects of low-frequency rTMS on in vitro and in vivo GBM models and to elucidate an underlying biological mechanism of rTMS on GBM. In vitro and in vivo GBM models were treated with low-frequency rTMS (0.5 ​Hz, 10 ​min per day), and the effects of rTMS were assessed using various assays, including CCK-8 assay, sphere formation assay, 3D invasion assay, RT-qPCR, Western blot, immunohistochemistry, TUNEL assay, MRI, and IVIS. The results showed that treatment of GBM models in vitro with low-frequency rTMS significantly inhibited cell proliferation. Transcriptome array analysis revealed a substantial downregulation of FLNA and FLNC expression after low-frequency rTMS treatment. Moreover, in an in vitro GBM tumor sphere model, low-frequency rTMS suppressed the activation of EGFR and EphA2, inhibited ERK/JNK/p38 and PI3K/AKT/mTOR pathways, and induced apoptosis. Low-frequency rTMS also suppressed the invasion of GBM by downregulating MMP2 and MMP9 expression. Additionally, in an in vivo GBM model, low-frequency rTMS suppressed GBM progression by downregulating FLNA and FLNC expression. The results demonstrated that low-frequency rTMS could be a potential treatment for GBM, achieved by downregulating FLNA and FLNC expression. This study sheds light on the potential for rTMS as a therapeutic strategy for glioblastoma as well as other types of cancers.

Keywords: Glioblastoma; Low-frequency; Repetitive transcranial magnetic stimulation; Tumor suppression effect.

Graphical abstract

Fig. 1

Low-frequency rTMS suppresses cell proliferation by downregulating the expression of FLNA and FLNC in the in vitro GBM model. U87MG were used as the in vitro GBM model. The model was divided into two groups: a sham group (non-treated, n ​= ​4) and a low-frequency group (treated with low-frequency rTMS, n ​= ​4). (A) Schematic figure of low-frequency rTMS treatment on an in vitro GBM model. (B) Cell counting kit-8 (CCK-8) assay of the in vitro GBM model with or without low-frequency rTMS treatment. (C) Quantification of CCK-8 assay. (D) ATP assay of in vitro GBM model with or without low-frequency rTMS treatment. (E) The relative gene expression of FLNA and FLNC in the in vitro GBM model with or without low-frequency rTMS treatment, as detected by RT-qPCR. (F) Western blot analysis of FLNA and FLNC in the in vitro glioblastoma model with or without low-frequency rTMS treatment. (G) Quantification of Western blot signals for FLNA and FLNC. Values are presented as means ​± ​standard error of the mean (SEM). Statistically significant differences are shown as ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 2

Low-frequency rTMS suppresses cell proliferation and sphereformation by downregulating FLNA and FLNC expression in in vitro GBM models. U87MG TS, TS15-88, and TS21-117 were used as the in vitro GBM models. Models were divided into two groups: a sham group (non-treated, n ​= ​4) and a low-frequency group (treated with low-frequency rMS, n ​= ​4). (A) In vitro GBM sphere models with or without low-frequency rTMS treatment. (B) The ratio of sphere formation in vitro with or without low-frequency rTMS treatment. (C) The sphere radius of in vitro GBM models with or without low-frequency rTMS treatment. (D) Cell counting kit-8 (CCK-8) assay of the in vitro GBM models with or without low-frequency rTMS treatment. (E) Quantification of CCK-8 assay. (F) ATP assay of in vitro GBM models with or without low-frequency rTMS treatment. (G) The relative gene expression of FLNA and FLNC in the in vitro GBM models with or without low-frequency rTMS treatment, as detected by RT-qPCR. (H) Western blot analysis of FLNA and FLNC in the in vitro GBM models with or without low-frequency rTMS treatment. (I) Quantification of Western blot signals for FLNA and FLNC. (J) Western blot analysis of FLNA and Ki-67 in the in U87MG TS transfected with FLNA or pCNV6 and with or without low-frequency rTMS treatment (K) Quantification of Western blot signals for FLNA and Ki-67. (L) Western blot analysis of FLNC and Ki-67 in U87MG TS transduced with FLNC or pCNV6 and with or without low-frequency rTMS treatment (M) Quantification of Western blot signals for FLNC and Ki-67. Values are presented as means ​± ​standard error of the mean (SEM). Statistically significant differences are shown as ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 3

Low-frequency rTMS downregulates the ERK/JNK/p38 and PI3K/AKT/mTOR pathway by suppressing EGFR and EphA2 activation in in vitro GBM models. (A) Western blot analysis of EGFR and EphA2 in the in vitro GBM models with or without low-frequency rTMS treatment. (B) Quantification of Western blot signals for EGFR and EphA2. (C) Western blot analysis of ERK, JNK, and p38 in the in vitro GBM models with or without low-frequency rTMS treatment. (D) Quantification of Western blot signals for ERK, JNK, and p38. (E) Western blot analysis of PI3K, AKT, and mTOR in the in vitro GBM models with or without low-frequency rTMS treatment. (F) Quantification of Western blot signals for PI3K, AKT, and mTOR. Values are presented as means ​± ​standard error of the mean (SEM). Statistically significant differences are shown as ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 4

Low-frequency rTMS induces apoptosis in the in vitro GBM models. (A) The relative gene expression of Bax and Bcl-2 detected by RT-qPCR. (B) Western blot analysis of Bax and Bcl-2 in the in vitro GBM models with or without low-frequency rTMS treatment. (C) Quantification of Western blot signals of Bax and Bcl-2. (D) TUNEL assay in the in vitro glioblastoma models with or without low-frequency rTMS treatment. (E) Quantification of TUNEL assay. Values are presented as means ​± ​SEM. Scale bars ​= ​100 ​μm. Statistically significant differences are shown as ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 5

Low-frequency rTMS suppresses invasion by downregulating the expression of MMP2 and MMP9 in the in vitro GBM models. (A) 3D invasion assay representing the invasive morphology of in vitro GBM models treated with or without low-frequency. (B) Quantification of 3D invasion assay. (C) The relative gene expression of MMP2 and MMP9 detected by RT-qPCR. (D) Western blot analysis of MMP2 and MMP9 in the in vitro GBM models with or without low-frequency rTMS treatment. (E) Quantification of Western blot signals for MMP2 and MMP9. Values are presented as means ​± ​standard error of the mean (SEM). Statistically significant differences are shown as ∗P ​< ​0.05, ∗∗∗P ​< ​0.001.

Fig. 6

Low-frequency rTMS suppressed tumor progression in an in vivo GBM model. The in vivo GBM model was divided into three groups: a sham group (non-treated), a low-frequency group (treated with low-frequency rTMS), and a TMZ group (treated with 30 ​mg/kg temozolomide). (A) Schematic figure of in vivo GBM model study. (B) MRI of brain tumor volume in sham, low-frequency, and TMZ groups (n ​= ​6). (C) Tumor progression of the in vitro GBM model with or without low-frequency rTMS treatment or TMZ, as measured by tumor volume in the brain from MRI. (D) Final tumor size of the in vitro GBM model with or without low-frequency rTMS treatment, or TMZ. (E) Bioluminescence images of tumor volume on the brain of sham, low-frequency, and TMZ groups (n ​= ​6). (F) Tumor progression of the in vitro GBM model with or without low-frequency rTMS treatment or TMZ, as measured by signal intensity of tumor mass in the brain. (G) Final signal intensity of tumor size in the in vitro GBM model with or without low-frequency rTMS treatment, or TMZ. (H) Survival rate for each group (n ​= ​4) was estimated based on Kaplan-Meier curves. Log-rank test (P ​= ​0.008). (I) Tumor mass stained by FLNA, FLNC and Ki67 antibody and H&E staining. (J) Quantification of cells stained with FLNA, FLNC and Ki67 antibody and H&E staining. (K) TUNEL assay in the in vivo GBM model with or without low-frequency rTMS treatment, or TMZ. (L) TUNEL assay quantification. (M) Tumor mass as stained by p-EphA, p-EGFR, p-ERK, p-JNK, p-p38, AKT, p-AKT, p-PI3K, p-mTOR, MMP2, and MMP9 antibody and H&E staining. (N) Quantification of cells stained with p-EphA, p-EGFR, p-ERK, p-JNK, p-p38, AKT, p-AKT, p-PI3K, p-mTOR, MMP2, and MMP9 staining. Values are presented as means ​± ​SEM. Scale bars ​= ​100 ​μm. Statistically significant differences are shown as ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.