Soloxolone para-methylanilide effectively suppresses aggressive phenotype of glioblastoma cells including TGF-β1-induced glial-mesenchymal transition in vitro and inhibits growth of U87 glioblastoma xenografts in mice

Abstract

Soloxolone amides are semisynthetic triterpenoids that can cross the blood-brain barrier and inhibit glioblastoma growth both in vitro and in vivo. Here we investigate the impact of these compounds on processes associated with glioblastoma invasiveness and therapy resistance. Screening of soloxolone amides against glioblastoma cells revealed the ability of compound 7 (soloxolone para-methylanilide) to inhibit transforming growth factor-beta 1 (TGF-β1)-induced glial-mesenchymal transition Compound 7 inhibited morphological changes, wound healing, transwell migration, and expression of mesenchymal markers (N-cadherin, fibronectin, Slug) in TGF-β1-induced U87 and U118 glioblastoma cells, while restoring their adhesiveness. Confocal microscopy and molecular docking showed that 7 reduced SMAD2/3 nuclear translocation probably by direct interaction with the TGF-β type I and type II receptors (TβRI/II). In addition, 7 suppressed stemness of glioblastoma cells as evidenced by inhibition of colony forming ability, spheroid growth, and aldehyde dehydrogenase (ALDH) activity. Furthermore, 7 exhibited a synergistic effect with temozolomide (TMZ) on glioblastoma cell viability. Using N-acetyl-L-cysteine (NAC) and flow cytometry analysis of Annexin V-FITC-, propidium iodide-, and DCFDA-stained cells, 7 was found to synergize the cytotoxicity of TMZ by inducing ROS-dependent apoptosis. Further in vivo studies showed that 7, alone or in combination with TMZ, effectively suppressed the growth of U87 xenograft tumors in mice. Thus, 7 demonstrated promising potential as a component of combination therapy for glioblastoma, reducing its invasiveness and increasing its sensitivity to chemotherapy.

Keywords: brain cancer; cancer stem cell; combination therapy; mesenchymal transition; triterpenoid.

FIGURE 1

Chemical structures of the soloxolone amides: N-(20-hydroxyethyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (1), N-(5′-hydroxypentyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (2), N-(2′-(dimethylamino)ethyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (3), N-(3′-(dimethylamino)propyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (4), N-(4′-bromophenyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (5), N-(pyridin-3-yl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (6), N-p-tolyl-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (7), N-(3′-(3″,5″-di-tert-butyl-4″-hydroxyphenyl)propyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30- amide (8), N-(2′-(1H-indol-2-yl)-ethyl)-2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-amide (9). The cyanoenone pharmacophore group, inherited by soloxolone amides from their predecessor soloxolone methyl (SM), is highlighted in red. The blue color indicates the position where the depicted amide moieties bind to the soloxolone scaffold.

FIGURE 2

Screening of the soloxolone amide library for GMT inhibitory activity. (A) The cytotoxicity of soloxolone amides against U87 cells assessed by MTT assay at 48 h (n = 4). The purple dotted line shows non-toxic concentrations causing less than 10% cell death (red dotted line). (B) Size distribution of U87 cells after treatment with TGF-β1 (50 ng/mL) and soloxolone amides (non-toxic concentrations) for 48 h (n = 3). (C) Adhesion of U87 cells to culture plastic after treatment with TGF-β1 (50 ng/mL) and soloxolone amides (non-toxic concentrations) for 48 h (n = 4). (D) Adhesion of U87 cells to Matrigel after treatment with TGF-β1 (50 ng/mL) and compound 7 (50 nM) for 48 h (n = 3). Data in line graph are represented as mean ± standard deviation (SD). Statistical significance was calculated by comparison with the control (marked by *) or TGF-β1-treated group (marked by ×). */×, **/××, ***/××× indicate that p-values were less than 0.05, 0.01, and 0.001, respectively.

FIGURE 3

Evaluation of GMT inhibition by compound 7. (A) Cytotoxicity of 7 against U118, EPNT-5 and hFF3 cells assessed by MTT assay at 48 h (n = 4). (B–G) The effect of 7 on the phenotypical manifestations of GMT was assessed by incubating U87 and U118 cells with TGF-β1 (50 ng/mL) and 7 (50 nM) for 48 h. (B) Adhesion of U118 cells to culture plastic measured by MTT assay (n = 4). (C) Shape distribution of U118 cells (n = 3). (D) Transwell migration of U87 cells (n = 4). (E) Wound closure on a monolayer of U118 cells (n = 3). (F, G) Expression of GMT markers in U87 (F) and U118 (G) cells assessed by RT-qPCR (n = 3). (H) Immunofluorescence staining of SMAD2/3 in U87 cells after pre-incubation with 7 for 48 h and activation with TGF-β1 for 1 h (I) SMAD2/3 distribution in U87 cells expressed as the ratio of nuclear (N) to cytoplasmic (C) intensity (n = 3, 100 cells/sample). (J) Binding energies of 7 to the kinase domains of TGF-β receptors type I and II (TβRI and TβRII) estimated using AutoDock Vina. For comparison, the affinities of known receptor inhibitors are given (PubChem CIDs 121411739 and 118988613 for TβRI and TβRII, respectively). (K) Docking structure of 7 with TβRI and TβRII visualized in ChimeraX. The orange and cyan dotted lines indicate hydrogen bonds and van der Waals interactions, respectively. Data in bar graphs are represented as mean ± standard deviation (SD). Statistical significance was calculated by comparison with the control or TGF-β1-treated group. *, **, *** indicate that p-values were less than 0.05, 0.01, and 0.001, respectively.

FIGURE 4

Effect of 7 on stemness of glioblastoma cells. (A–C) Colony forming activity of U87 (A), U118 (B) and EPNT-5 (C) cells assessed after 10 days of incubation with 7 (0–1,000 nM) (n = 5). (D) Growth of U87 primary tumorspheres over 10 days of 7 treatment (0–1,000 nM) (n = 8). (E) Size of U87 secondary tumorspheres that were formed from cells of primary tumorspheres after treatment with 7 (0–1,000 nM) for 4 days (n = 8). (F) ALDH activity in U87 primary tumorspheres after 4 days of incubation with 7 (0–100 nM) for 4 days assessed using AldeRed staining and flow cytometry (n = 3, 10⁴ events/sample). Line graph data are represented as mean ± standard deviation (SD). Statistical significance was calculated by comparison with the untreated group. *, **, *** indicate that p-values were less than 0.05, 0.01, and 0.001, respectively.

FIGURE 5

Evaluation of the synergistic effect of 7 and TMZ against glioblastoma cells. (A, B) Apoptosis and necrosis assessed in U87 (A) and U118 (B) cells after 48 h of incubation with 7 (0–2000 nM) using Annexin V-FITC and propidium iodide double staining and flow cytometry (n = 3, 10⁴ events/sample). (C) Cytotoxicity of combinations of 7 (0–400 nM) and TMZ (0–500 μM) against U87, U118 and EPNT-5 cells evaluated by MTT assay (n = 4). (D) Synergy score (δ) across different concentrations of 7 and TMZ in U87, U118 and EPNT-5 cells (n = 4). δ was calculated using the HSA model in the SynergyFinder + platform. Black crosses indicate maximum δ values. (E) The impact of ROS scavenging by NAC (2 mM) on the combined cytotoxicity of TMZ (125 μM) and 7 (100 nM) in U87, U118, and EPNT-5 cells (n = 4). Incubation time: 72 h (F) ROS production in U87 cells after 72 h of incubation with TMZ (125 μM) and 7 (100 nM) assessed by DCFDA staining and flow cytometry (n = 3, 10⁴ events/sample). (G) Apoptosis and necrosis assessed in U87 cells after 72 h of incubation with TMZ (500 μM), 7 (600 nM), and NAC (2 mM) using Annexin V-FITC and propidium iodide double staining and flow cytometry (n = 3, 10⁴ events/sample). Data in line graphs, bar graphs, and lollipop chart are represented as mean ± standard deviation (SD). Statistical significance was calculated by comparison with control (indicated by *) or the corresponding NAC-treated group (indicated by ×). */×, **/××, ***/××× indicate that p-values were less than 0.05, 0.01, and 0.001, respectively.

FIGURE 6

Antitumor effect of compound 7 and its combination with TMZ on U87 glioblastoma xenograft model. (A) Experimental setup. U87 glioblastoma cells were subcutaneously (s.c.) implanted into athymic nude mice. On day 4 after tumor transplantation mice were treated with compound 7 (20 mg/kg, i.p., n = 5), TMZ (10 mg/kg, i.p., n = 5) or their combination at the same doses and administration route (n = 6). Compound 7 was administered three times per week for a total of seven injections. TMZ was administered daily, except weekends, for a total of eleven injections. Vehicle-treated mice were used as control (n = 6). Mice were sacrificed on day 19 after tumor transplantation, and material (tumor nodes, livers, and kidneys) was collected for further analysis. (B–D) Compound 7 effectively suppresses the growth of U87 glioblastoma. Tumor volumes (B), tumor weights (C), and primary tumor nodes (D) of U87 glioblastoma without treatment and after administration of 7, TMZ or their combination. The insert shows tumor volumes on day 13 of tumor growth. (E, D) Mice weights (E) and relative organ indices (F) of U87 glioblastoma-bearing mice without treatment and after administration of 7, TMZ or their combination.

FIGURE 7

Histological structure of U87 glioblastoma without treatment and after administration of 7, TMZ or their combination. (A) Representative histological images of U87 glioblastoma in control and experimental groups. The black boxes show areas that were examined further at a higher magnification. Hematoxylin and eosin staining. Original magnification ×200 (left panel) and ×400 (right panel). (B) The numerical density (Nv) of tumor cells in the state of mitosis (n = 5–6). (C) Immunohistochemical staining of U87 glioblastoma with primary anti-Ki-67 antibodies. The black arrows indicate Ki-67 positive cells. Original magnification ×400. (D) The numerical density (Nv) of Ki-67 positive cells (n = 3).

FIGURE 8

The effect of 7, TMZ or their combination on the expression of N-cadherin and GFAP in U87 glioblastoma. (A) Survival of patients with recurrent glioma depending on the expression levels of CDH2 and GFAP (CCGA database). (B) Expression levels of CDH2 and GFAP in mesenchymal- and proneural-type glioblastoma (REMBRANDT database). *** indicates that p-values were less than 0.001. (C) Representative images of U87 glioblastoma immunohistochemically stained with primary anti-N-cadherin (upper panel) and anti-GFAP (lower panel) antibodies. Original magnification ×400.