The therapeutic value of XL388 in human glioma cells

Abstract

XL388 is a highly efficient and orally-available ATP-competitive PI3K-mTOR dual inhibitor. Its activity against glioma cells was studied here. In established and primary human glioma cells, XL388 potently inhibited cell survival and proliferation as well as cell migration, invasion and cell cycle progression. The dual inhibitor induced significant apoptosis activation in glioma cells. In A172 cells and primary human glioma cells, XL388 inhibited Akt-mTORC1/2 activation by blocking phosphorylation of Akt and S6K1. XL388-induced glioma cell death was only partially attenuated by a constitutively-active mutant Akt1. Furthermore, it was cytotoxic against Akt1-knockout A172 glioma cells. XL388 downregulated MAF bZIP transcription factor G (MAFG) and inhibited Nrf2 signaling, causing oxidative injury in glioma cells. Conversely, antioxidants, n-acetylcysteine, pyrrolidine dithiocarbamate and AGI-106, alleviated XL388-induced cytotoxicity and apoptosis in glioma cells. Oral administration of XL388 inhibited subcutaneous A172 xenograft growth in severe combined immunodeficient mice. Akt-S6K1 inhibition and MAFG downregulation were detected in XL388-treated A172 xenograft tissues. Collectively, XL388 efficiently inhibits human glioma cell growth, through Akt-mTOR-dependent and -independent mechanisms.

Keywords: Akt; MAFG; XL388; glioma; mTOR.

Figure 1

XL388 potently inhibits glioma cell survival, proliferation, migration, invasion and cell cycle progression. A172 cells (A–H), U251MG cells (“U251”) (I–K) and primary human glioma cells (“Pri-1/Pri-2”) (I–K) were treated with applied concentrations of XL388 or the vehicle control (“C”, same for all Figures), and cultured for applied time periods, then cellular functions including cell survival (A, B and I), proliferation (C–E, and J), migration (F and K), invasion (G) and cell cycle progression (H) were tested by the indicated assays. Results were quantified. Expression of listed proteins was shown (A). Data were presented as mean ± SD (n=5). * p <0.05 vs. “C” cells. Experiments in this figure were repeated three times, and similar results were obtained. Bar= 100 μm (D, F and G).

Figure 2

XL388 induces significant apoptosis activation in glioma cells. A172 cells (A–F), U251MG (“U251”) (G and H) and primary human glioma cells (“Pri-1/Pri-2”) (G and H) as well as the primary human astrocytes (“Astrocytes”) and HCN-1a neuronal cells (I and J) were treated with XL388 (250 nM), and cultured for applied time periods, then cell apoptosis was analyzed by the mentioned assays (A–H and J), with cell viability tested by CCK-8 assay (I). Data were presented as mean ± SD (n=5). * p <0.05 vs. “C” cells. Experiments in this figure were repeated three times, and similar results were obtained. Bar= 100 μm (D and E).

Figure 3

XL388-induced anti-glioma cell activity is through Akt-mTOR-dependent and -independent mechanisms. A172 cells or the primary human glioma cells, Pri-1, were treated with XL388 (250 nM), and cultured for 2h, and expression of listed proteins was shown (A and B). A172 cells or the Pri-1 primary human glioma cells were treated with XL388 (250 nM), LY294002 (“LY”, 1 μM), perifosine (“Prf”, 5 μM) or rapamycin (“Rap”, 500 nM) for 48-72h, then cell viability and apoptosis were tested by CCK-8 (C and D) and TUNEL staining (E and F) assays, respectively. Stable A172 cells with the CRISPR/Cas9-Akt1-KO construct (“Akt1-KO” cells) or empty vector (“Vec”) were treated with or without XL388 (250 nM) for applied time, and cultured for applied time periods, and expression of listed proteins was shown (G);Cell death and apoptosis were tested by Trypan blue staining (H) and nuclear TUNEL staining (I) assays, respectively. Stable A172 cells with a constitutive-active Akt1 (S473D, “ca-Akt1”) or empty vector (“Vec”) were treated with or without XL388 (250 nM) for applied time, and expression of listed proteins was shown (J); cell death and apoptosis were tested by Trypan blue staining (K) and nuclear TUNEL staining (L) assays, respectively. Data were presented as mean ± SD (n=5).* p <0.05 vs. “C” cells. ^#p <0.05 vs. XL388 treatment (C–F). ^#p <0.05 (H, I, K and L). Experiments in this figure were repeated three times, and similar results were obtained.

Figure 4

XL388 induces oxidative injury in human glioma cells. A172 cells or primary human glioma cells (“Pri-1”) were treated with XL388 (250 nM) and cultured for indicated time periods, then expression of listed mRNAs and proteins was tested by qPCR and Western blotting assays (A–C); Relative CellROX intensity (D) and lipid peroxidation (E) levels were tested. A172 cells were pretreated for 1h with n-acetylcysteine (NAC, 400 μM), pyrrolidine dithiocarbamate (PDTC, 10 μM) or AGI-1067 (10 μM), followed by XL388 (250 nM) stimulation for another 48-72h, then cell viability and apoptosis were tested by CCK-8 (F) and nuclear TUNEL staining (G) assays, respectively. U251MG (“U251”) and primary human glioma cells (“Pri-1/Pri-2”) were treated with XL388 (250 nM) for 12h, then the relative CellROX intensity was tested (H). A172 cells with the CRISPR/Cas9-Akt1-KO construct (“Akt1-KO” cells) or empty vector (“Vec”) were treated with or without XL388 (250 nM) and expression of listed proteins was shown (I). Relative ROS contents were tested by measuring CellROX intensity (J). Expression of MAFG protein in A172 cells and primary human astrocytes (“Astrocytes”) was shown (K); Astrocytes were treated with or without XL388 (250 nM) for 24h, and ROS intensity tested by CellROX assay (L). Data were presented as mean ± SD (n=5).* p <0.05 vs. “C” cells. ^#p <0.05. “Veh”-pretreated cells (F, G and J). Experiments in this figure were repeated three times, and similar results were obtained. Bar= 100 μm (D).

Figure 5

XL388 oral administration inhibits A172 xenograft growth in SCID mice. The SCID mice bearing A172 xenografts (n=10 per group) were administrated with vehicle (saline, “Veh”) or XL388(5 mg/kg body weight, daily, × 14d), then tumor volumes (in mm³) (A) and mice body weights (in grams) (D) was recorded every seven days for a total of 35 days; The estimated daily tumor growth (in mm³ per day) was calculated as described (B). At treatment Day-35, all tumors were isolated and individually weighted (C). At treatment Day-7, two hours after initial XL388administration, the xenograft tumors were isolated. Tissue lysates were subjected to Western blotting assays of listed proteins (E and F). *p < 0.05 vs. “Veh” group (A–C).