Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Abstract

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.

FIGURE 1.

A cell-based screen for inhibitors preventing the cell proliferation of the most malignant U87MG/EGFRvIII versus less severe U87MG/PTEN cells. The diagram of drug screening strategy. U87MG/EGFRvIII cells were cultured in 96-well plates and treated with 2.5 μm compounds from chemical libraries for 4 days followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The compounds with more than 70% cell growth inhibitory effect were considered positive hits. The positive compounds from this round were subjected to U87MG/PTEN cells. The compounds that displayed <40% inhibition on cell proliferation were picked for the cytotoxicity assay on non-cancerous HEK293 cells and mouse embryonic fibroblast cells. Only compounds that exhibited negligible toxicity on these cells were selected.

FIGURE 2.

Validation and SAR study of acridine yellow G. A, cell proliferation assay. A SAR study was carried out on U87MG/EGFRvIII and U87MG/PTEN cells. Numerous tricyclic compounds were selected and analyzed on these two cell lines for their anti-proliferative activity. U87MG/EGFRvIII and U87MG/PTEN cells were treated with different compounds (1 μm) having similar structures for 96 h. Data were expressed as mean ± S.E. Acridine yellow G exhibited an evident inhibitory effect on U87MG/EGFRvIII, but only weakly blocked U87MG/PTEN cell growth. The central ring is essential for the anti-proliferative effect. The two amino groups can be modified by small alkyl groups and they can also be switched with methyl groups on the side rings. B, chemical structures of acridine derivatives.

FIGURE 3.

Acridine yellow G blocks EGFR/PKC/mTOR signaling pathways. A, in vitro kinase assay. Acridine yellow G inhibited EGFR kinase activity but had no effect on mTOR kinase activity in vitro. Purified human EGFR kinase or rat mTOR kinase were incubated with different concentrations of acridine yellow G on ice for 20 min and the mixture was added to the respective substrate-coated wells. The phosphorylated substrates were detected by anti-phosphotyrosine or anti-phospho-p70S6K 389 for EGFR kinase and mTOR kinase, respectively. The kinase assays were performed in triplicate. Data were expressed as mean ± S.E. B, in vitro PKC kinase assay. Acridine yellow G inhibited different PKC kinases in a dose-dependent manner. Acridine yellow G was subjected to the SelectScreen Kinase Profiling Panel analysis using Z′-LYTE substrates. The kinase activities of PKCα, PKCβ, and PKCγ were totally suppressed by 10 μm acridine yellow G. C, acridine yellow G suppresses EGFR, mTOR, and PKC signaling pathways in GBM cells. U87MG/EGFRvIII and U87MG/PTEN cells were treated with different concentrations of acridine yellow G for 6 h and the cells were lysed. The lysates were analyzed by immunoblotting (IB) with various antibodies as indicated. EGFR tyrosine phosphorylation and numerous mTOR downstream effectors' activation were blocked by acridine in a dose-dependent manner. Acridine yellow G also suppressed PKC phosphorylation. D, cell proliferation assay. Acridine yellow G preferentially blocked the cell proliferation of U87MG/EGFRvIII cells in a dose-dependent manner. The IC₅₀ is about 0.25 μm. Data were expressed as mean ± S.E.

FIGURE 4.

Acridine yellow G arrests cell cycle at G₁ phase. A, the expression profiles and phosphorylation of PKCs in U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in all relevant combinations. Cells that overexpress PTEN showed higher PKC protein expression levels as well as stronger phosphorylation than the counterpart without PTEN. B, acridine yellow G arrests the cell cycle at the G₁ phase. U87MG, U87MG/PTEN, U87MG/EGFR, and U87MG/EGFRvIII cells were treated with acridine yellow G (1 μm) for 24 h. Flow cytometric analysis showed that acridine yellow G induced cell cycle arrest at G₁ in the 4 tested isogenic U87MG cell lines. C, flow cytometry analysis of the effect of PKC inhibitors in combination with EGFR inhibitor erlotinib. Four different U87MG isogenic cell lines were incubated with 100 nm of different PKC inhibitors in the presence or absence of erlotinib (10 μm) for 24 h. The treated cells were analyzed by flow cytometry. PKC inhibitor had a negligible effect on cell cycle profiles. D, knocking down the PKCs had no effect on the acridine yellow G G₁ phase arrest effect. PKCα and PKCβI were depleted by its specific siRNA in U87MG/EGFRvIII and U87MG/EGFRvIII/PTEN cells. After 24 h, the siRNA-treated cells were further treated with 1 μm acridine for another 24 h, followed by flow cytometry analysis.

FIGURE 5.

Acridine yellow G blocks subcutaneous tumor growth of U87MG/EGFRvIII cells. A and B, acridine yellow G significantly inhibits the growth of U87MG/EGFRvIII xenograft tumors. U87MG/EGFRvIII cells were inoculated subcutaneously in nude mice, and after the tumors formed, the nude mice were treated orally with vehicle or acridine yellow G at doses of 50 and 100 mg/kg, respectively. Control mice were treated with vehicle (0.5% methylcellulose). The drug and vehicle control were administrated via oral gavage. Data represent mean ± S.E. (*, p < 0.05; **, p < 0.01, n = 11–12/group). C and D, acridine yellow G inhibits EGFR, mTOR, and PKC signalings in vivo. C, the tumor tissue lysates from vehicle or drug-treated samples were analyzed by immunoblotting (IB) with the indicated antibodies. EGFR tyrosine phosphorylation and numerous mTOR downstream effectors' activation were blocked by C2. Moreover, C2 also inhibited PKC phosphorylation but had no effect on Akt signaling. D, immunohistochemistry for p-EGFR (Y1068), p-Akt (S473), p-PKC (pan), and p-rpS6 (S235/236) on tumor sections derived from animals treated with or without acridine yellow G. Brown represents positive staining (arrows). Bar represents 100 μm. E, quantification of p-EGFR expression level versus the total EGFR in subcutaneous tumor. Data represent mean ± S.E. (**, p < 0.01). F and G, acridine yellow G inhibits tumor growth in vivo. Ki67 (F) and active caspase-3 (G) staining were conducted on tumor sections derived from animals treated with or without acridine yellow G. Bar represents 50 μm.

FIGURE 6.

Acridine yellow G suppresses intracranial tumor growth of U87MG/EGFRvIII cells. A and B, acridine yellow G suppresses the growth of U87MG/EGFRvIII xenograft tumors in brain. A, MRI scans of individual mice 14 days after intracranial implantation of 10⁵ tumor cells. The presence of a glioma is detected through the bright areas (red arrows) by contrast enhancement from the MRI contrast agent (Gd-DTPA). U87MG/EGFRvIII brain tumor growth in brains of mice was reduced by orally administrated acridine yellow G. Note the small tumors in acridine yellow G-treated mice and the large tumor in vehicle-treated animals. B, quantitative analysis of intracranial tumor volume in mice treated with or without the compound. Acridine yellow G significantly suppressed tumor growth compared with the control group. Data represent mean ± S.E. (*, p < 0.05, n = 12/group). C and D, acridine yellow G represses EGFR/PKC/mTOR signalings in brain tumors. C, Western blot (IB) analysis of intracranial tumor lysates from the animals treated with acridine yellow G (50 and 100 mg/kg) or vehicle for 15–20 days. EGFR tyrosine phosphorylation and numerous mTOR downstream effectors' activation were blocked by C2. D, immunohistochemistry for p-EGFR (Y1068), p-Akt (S473), p-PKC (pan), and p-rpS6 (S235/236) in tumor sections derived from animals treated with or without acridine yellow G. Brown represents positively stained cells (arrows). Bar represents 100 μm. E and F, acridine yellow G inhibits intracranial tumor growth. Ki67 (E) and active-caspase-3 (F) staining were conducted on tumor sections derived from animals treated with or without acridine yellow G. Bar represents 50 μm. G, survival curves of brain tumor-bearing mice. The nude mice were implanted stereotactically in the brain. After confirmation of brain tumor formation by MRI, the mice were orally treated with or without C2 once a day until death. The acridine yellow G treatment group showed a significant improvement in their survival compared with the vehicle treatment group (p < 0.05). H, quantification of p-EGFR level versus the total EGFR in intracranial tumor. Data represent mean ± S.E. (**, p < 0.01)

FIGURE 7.

Acridine yellow G displays no neurotoxicity in vivo. A, acridine yellow G exhibits no neurotoxicity. Histological analysis as well as anti-caspase-3 and TUNEL staining were carried out on the adjacent neural tissues of intracranial model animals receiving C2 and vehicle. Compared with the vehicle treatment group, C2 exerts no neurotoxicity or neuronal damage. B, acridine yellow G does not induce apoptosis at 1 μm. U87MG, U87MG/PTEN, U87MG/EGFR, and U87MG/EGFRvIII cells were treated with C2 (1 μm) for the indicated times. The cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. C, acridine yellow G triggers apoptosis at higher doses. U87MG/PTEN and U87MG/EGFRvIII cells were treated with the indicated doses of C2 for 24 h. A high dose of C2 (5 and 10 μm) induced clear apoptosis in cells.