Therapeutic potential of valsartan in overcoming chemoresistance in human glioblastoma

Figures

• 

  Figure 1.. Valsartan decreased the proportion of viable TMZ-S and TMZ-R GBM cells in a dose-dependent manner. (A) TMZ-resistant glioma clones were established through drug exposure. An MTS assay was used to determine the viability of the indicated cells treated with TMZ (0–400 μM for 48 h; n = 3). (B) (Left panel) Effects of treatment with different concentrations of valsartan for 48 h (0–400 µM, n = 3) on the viability of nontumor control cells (NHAs) and TMZ-S and TMZ-R LN229 and GBM8401 clones assessed via MTS assays. Gray represents normal NHAs, red represents LN229 cells, light blue represents LN229 TMZ-R cells, yellow represents GBM8401 cells, light green represents GBM8401 TMZ-R cells, and the black dotted line indicates the IC50. (Right panel) The IC50 value at 48 h was determined from the dose−response curve. (C) Expression of MGMT protein in TMZ-S and TMZ-R glioma cells treated with valsartan at the indicated dose, analyzed by western blotting. Representative images from two independent tests are shown.

• 

  Figure 2.. Valsartan dose-dependently inhibited cell proliferation. Cells were stained with BrdU and analyzed by flow cytometry to measure proliferation of LN229 TMZ-S (A), LN229 TMZ-R (B), GBM8401 TMZ-S (C), and GBM8401 TMZ-R (D) clones (0, 100, 200, and 300 μM, respectively, for 48 h; unpaired two-tailed Student's t test; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001).

• 

  Figure 3.. Valsartan inhibited colony formation in a dose-dependent manner. (A–D) Colony formation of LN229 TMZ-S (A), LN229 TMZ-R (B), GBM8401 TMZ-S (C), and GBM8401 TMZ-R (D) cells treated with 0, 100, 200, or 300 μM valsartan for 14 days (unpaired two-tailed Student's t test; n = 3; **p < 0.01, ***p < 0.001).

• 

  Figure 4.. Valsartan-induced apoptosis in TMZ-S and TMZ-R glioma cells. (A–D) Apoptotic proportions of LN229 TMZ-S (A), LN229 TMZ-R (B), GBM8401 TMZ-S (C), and GBM8401 TMZ-R (D) clones treated with 0, 100, 200, or 300 μM valsartan, analyzed by flow cytometry based on annexin V and 7-AAD positivity. Bars indicate means ± SDs (unpaired two-tailed Student's t test; n = 3; **p < 0.01, ***p < 0.001). (E–H) Western blots showing γ-H2AX and survivin levels in LN229 TMZ-S (E), LN229 TMZ-R (F), GBM8401 TMZ-S (G), and GBM8401 (H) TMZ-R glioma cells treated with 0, 100, 200, and 300 μM valsartan for 48 h. Bars indicate means ± SDs (unpaired two-tailed Student's t test; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

• 

  Figure 5.. Valsartan inhibited the migration capacity of TMZ-S and TMZ-R glioma cells in a dose-dependent manner. (A–D) The migration capacity of LN229 TMZ-S (A), LN229 TMZ-R (B), GBM8401 TMZ-S (C), and GBM8401 TMZ-R (D) clones was analyzed via a wound healing assay. (E–H) Expression of EMT marker snail in TMZ-S and TMZ-R glioma cells was assessed by western blotting (0–300 μM, for 48 h; unpaired two-tailed Student's t test; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001).

• 

  Figure 6.. Valsartan contributed to cell cycle arrest at the G1 phase in TMZ-S and TMZ-R glioma cells. Cell cycle analysis of LN229 TMZ-S cells. (A), LN229 TMZ-R (B), GBM8401 TMZ-S (C), and GBM8401 TMZ-R (D) clones were analyzed by flow cytometry via PI staining (0–300 μM, for 48 h; 6n = 3).

• 

  Figure 7.. APLN is a potential downstream target of valsartan. (A) RNA sequencing revealed the top 10 upregulated and downregulated genes. (B–E) APLN mRNA expression in LN229 TMZ-S (B), LN229 TMZ-R (C), GBM8401 TMZ-S (D), and GBM8401 TMZ-R (E) clones was measured via RT−PCR after treatment with valsartan (0–300 μM, for 48 h, unpaired two-tailed Student's t test, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). Expression levels of APLN in different stages of glioma tissue were analyzed via GEPIA (F) and GENT2 (G). Kaplan–Meier survival analysis was performed using data from GEPIA (H) and the Chinese Glioma Genome Atlas (CGGA) (I). T: tumor; N: normal control; LGG: low-grade glioma; HR: hazard ratio; p(HR): pooled hazard ratio; TPM: transcripts per million. TMZ: temozolomide; GEPIA: Gene Expression Profiling Interactive Analysis; GENT2: Gene Expression Database of Normal and Tumor Tissues 2.