Anlotinib combined with temozolomide suppresses glioblastoma growth via mediation of JAK2/STAT3 signaling pathway

Abstract

Purpose: Anlotinib protects against carcinogenesis through the induction of autophagy and apoptosis. The current study evaluated the role and molecular mechanisms of anlotinib in glioblastoma, and the effects of anlotinib in combination with temozolomide (TMZ).

Methods: Cell Counting Kit-8 and colony-forming assays were used to evaluate cell viability. Cell migration and invasion were assessed by wound-healing, Transwell migration, and Matrigel invasion assays. Cellular apoptosis and cell cycle analysis were determined by flow cytometry. Angiogenesis was assessed using human umbilical vein endothelial cells (HUVECs). Vascular endothelial growth factor A (VEGFA) was measured by enzyme-linked immunosorbent assay. Protein expression was determined by western blotting or immunofluorescence staining. The in vivo anti-glioblastoma effect was assessed with live imaging of tumor xenografts in nude mice.

Results: Anlotinib restricted the proliferation, migration, and invasion of glioblastoma cells in a dose-dependent manner. Tumor supernatant from glioblastoma cells treated with anlotinib inhibited angiogenesis in HUVECs. Anlotinib induced autophagy in glioblastoma cells by increasing Beclin-1 and microtubule-associated protein 1 light chain 3B (LC3B) levels. Mechanistically, anlotinib inhibited the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3)/VEGFA signaling pathway. STAT3 inhibition by S3I-201 decreased VEGFA and suppressed cellular proliferation and movement. TMZ enhanced the anti-glioblastoma ability of anlotinib. Finally, anlotinib inhibited tumor growth and JAK2/STAT3/VEGFA signaling in xenografts.

Conclusion: Anlotinib exerts anti-glioblastoma activity possibly through the JAK2/STAT3/VEGFA signaling pathway. TMZ potentiated the anti-glioblastoma effect of anlotinib via the same signaling pathway, indicating the potential application of anlotinib as a treatment option for glioblastoma.

Keywords: Anlotinib; Glioblastoma; Invasion; JAK2/STAT3/VEGFA signaling pathway; Migration; Proliferation.

Fig. 1

Anlotinib inhibits the proliferation of glioblastoma cells. a–c Anlotinib suppressed the growth of human glioblastoma cell lines (A172, U87, and U251) in both a dose- and time-dependent manner. d Representative images of the colony-formation assay. Cells were incubated for 14 days with increasing concentrations of anlotinib and colony formations were reduced in all tested human glioblastoma cell lines. e Quantification of the colony-formation ability. The values are expressed as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control

Fig. 2

Anlotinib suppresses the migration and invasion capacity of glioblastoma cells in vitro. a A172, U251 and U87 cells were treated with vehicle or anlotinib for 24 h. Cells that migrated into the wounded areas were observed and imaged. b Quantification of the wound-healing ability. c, e The migration and invasion of glioblastoma cells in Transwell migration and Matrigel invasion assays were inhibited. d, f Quantification of the number of migrated and invasive cells was analyzed from five independent fields of view. The wound-healing ability of the vehicle-treated group was adjusted to the value of 100. Values are expressed as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control

Fig. 3

Anlotinib induces G2/M phase cell cycle arrest, induces apoptosis, mediates expression of apoptosis-related proteins, and triggers autophagy in glioblastoma cells. a, b U87 cells were tested in after anlotinib treatment for 24 h by flow cytometry and arrested at the G2/M phases. c, d Anlotinib enhanced the ratio of apoptotic cells in a dose-dependent manner. e, f Anlotinib upregulated the levels of pro-apoptotic proteins (BAX and cleaved caspase-3). In addition, anlotinib downregulated the level of the anti-apoptotic protein Bcl-2. The cells were pretreated with anlotinib for 24 h. g, h Anlotinib reduced the expression levels of Beclin-1 and LC3B as detected by western blotting. i Immunofluorescence staining of LC3B showed that anlotinib increased autophagosome formation, and this process was suppressed by an inhibitor of autophagy (3-MA). Data are represented as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs control group

Fig. 4

Anlotinib inhibits vascularization through the JAK2/STAT3/VEGFA pathway. a Representative images of tubular formation assay. HUVECs were treated with corresponding tumor supernatant and subjected to a tubular formation assay. b Quantification of the number of vessels was analyzed using data from three independent tests. c The levels of VEGFA in the tumor supernatant from U87 cells were measured by ELISA. d, e, f Western blotting showed that decreased expression of p-JAK2, STAT3, p-STAT3, and VEGFA after anlotinib treatment. In addition, the anlotinib-effect on STAT3, p-STAT3, and VEGFA was enhanced by S3I-201. g, h The decreased expression of cell cycle phase-related proteins (cyclin A2 and cyclin D1) and cell motility-related proteins (HMGB1 and MMP2) by anlotinib were potentiated by S3I-201. The relative protein levels in control cells were adjusted to the value of 1. Data are represented as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs control group, ^#p < 0.05, ^##p < 0.01 vs anlotinib group

Fig. 5

The combination of anlotinib and TMZ enhances the effect of anti-glioblastoma cells. a The combination of anlotinib and TMZ enhanced the effect of suppressing growth of human glioblastoma cell lines (U87 and U251), compared with single drug treatments. b, c Anlotinib combined with TMZ greatly suppresses the migration capacity of glioblastoma cells. d, e The combination treatment enhanced the cytotoxic effects in human glioblastoma cell lines. f, g The suppressive effect on the JAK2/STAT3/VEGFA pathway was enhanced with the combination of drugs. The relative protein levels in control cells were adjusted to the value of 1. Data are represented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 vs anlotinib group

Fig. 6

Anlotinib restraints GBM growth in vivo. a Representative bioluminescent images showed tumor volumes in orthotopic GBM model at 7, 14 and 28 days. b Quantification of tumor volumes in the different treatment arms. c Representative images of IHC staining of Ki-67. d Tumor tissues from two different nude mice in each treatment group were analyzed by western blotting. The relative tumor volumes of the control group were adjusted to the value of 1. Data were represented as means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group