Two cell lines were used in this study. The T98G human glioma tumor cell line was purchased from ATCC (Manassas, VA, United States); this is a naturally TMZ-resistant cell line with elevated levels of MGMT. The U251/TMZ cell line was a gift from Dr. Yuhui Zou of General Hospital of Guangzhou Military Command of People’s Liberation Army, as previously reported (Ji et al., 2018); this line has acquired TMZ resistance, with overactivated hedgehog signaling. Both cell lines were cultured in Dulbecco’s modified eagle medium (Gibco, TX, United States) with 10% (v/v) fetal bovine serum (Gibco), penicillin (100 units/ml), and streptomycin (100 units/ml) in a humidified atmosphere of 5% CO₂ at 37°C.

XH30 was synthesized in house as described previously (Lin et al., 2018). PI3K inhibitor PF-04691502 was purchased from Selleck Chemicals (Houston, TX, United States). TMZ was purchased from J&K Scientific (Beijing, China). All antibodies [AKT, phosphor-AKT (S473), phosphor-AKT (T308), mammalian target of rapamycin (mTOR), phosphor-mTOR (S2448), GSK3β, phosphor-GSK3β (S9), proline-rich AKT substrate of 40 kDa (PRAS40), phosphor-PRAS40 (T246), p70S6K, phosphor-p70S6K (T389), S6 ribosomal protein (S6RP), phosphor-S6RP (S240/244), smoothened (SMO), cyclin D1, and anti-cyclin–dependent kinase (CDK2)] were purchased from Cell Signaling Technology (Danvers, MA, United States). Anti-GLI1 and anti–β-actin antibodies were purchased from Abcam (Cambridge, United Kingdom) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Solarbio, Beijing, China). Briefly, 2,000 cells per well were seeded into a 96-well plate. After incubation overnight, the cells were treated with different concentrations (0.0512, 0.256, 1.28, 6.4, 32, 160, 800, 4,000, and 20,000 nM) of XH30 or PF-04691502 with three replicates for 72 h. Then, CCK-8 solution was added and incubated for 3 h, after which, absorbance values were measured at 450 nM using a microplate reader (BioTek Instruments, Inc., United States). The half-maximal inhibitory concentration (IC₅₀) was calculated using GraphPad Prism v8.0.1 (La Jolla, CA, United States).

U251/TMZ and T98G cells were seeded at a density of 200 cells per well into six-well plates. After 24 h, the cells were treated with indicated concentrations (4, 20, 100, and 500 nM) of XH30 with three replicates. The culture medium with test compound was replaced every 3 days. After cell colonies formed, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min, then stained with crystal violet for 30 min, and washed with PBS. Finally, colonies were recorded with a photograph and measured by a microplate reader (BioTek).

Flow cytometry assays were used to analyze the cell cycle distribution as previously reported (Ji et al., 2018). In brief, U251/TMZ and T98G cells were dispensed into six-well plates at a density of 50,000 cells per well. After growing overnight in a humidified atmosphere of 5% CO₂ at 37°C, cells were treated with indicated concentrations (4, 20, 100, and 500 nM) of XH30 for 24 h. Then, cells were harvested and fixed ice cold 70% ethanol overnight at –20°C, washed with PBS, and stained with propidium iodide (PI) solution containing PI (20 mg/ml) and RNase A (20 mg/ml) in PBS for 30 min. DNA contents were measured using the BD fluorescence-activated cell sorting verse flow cytometer (BD Biosciences, NJ, United States), and the cell cycle distribution was analyzed.

Cells or mice tumor tissues were collected and lysed in RIPA lysate buffer supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Solarbio, Beijing, China). Lysates were then centrifuged at 12,000 g for 30 min. Proteins were quantified using a bicinchoninic acid assay kit (Solarbio, Beijing, China). Resultant samples containing equal amounts of proteins were subjected to sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was blocked with TBST buffer containing 5% non-fat milk for 30 min and incubated with appropriate primary antibodies (1:1,000 dilution) in TBST at 4°C overnight. After washing with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies (1:2,000 dilution; Cell Signaling Technologies, Boston, MA) for 1 h at room temperature. Bound proteins were visualized using enhanced chemiluminescence and detected using ImageQuant LAS 4000 software.

Total RNA from XH30-treated U251/TMZ or T98G cells was isolated using TRIzol reagent (Bioteke Corporation, China) according to the recommended procedures of the manufacturer. First-strand cDNA was synthesized from 1 μg of total RNA using the ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (Toyobo, Japan). Real-time polymerase chain reaction (PCR) was performed using the Analytikjena qTOWER detection system and the SYBR^® Green RT-PCR master mix (Toyobo). Target sequences were amplified at 95°C for 1 min, followed by 40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. Fold changes in GLI1, paired box protein 6 (PAX6), and O6-methylguanine-DNA-MGMT (MGMT) gene expression were calculated according to the 2−ΔΔCt method. The primers sequences used to amplify specific regions of the indicated genes were as follows: GLI1 forward, ATG​TTC​AAC​TCG​ATG​ACC​CCA​C; GLI1 reverse, CAA​CTT​GAC​TTC​TGT​CCC​CAC​A; MGMT forward, ATGGAT GTTTGAGCGACACA; MGMT reverse, ATA​GAG​CAA​GGG​CAG​CGT​TA; PAX6 forward, AAC​GAT​AAC​ATA​CCA​AGC​GTG​T; PAX6 reverse, GGT​CTG​CCC​GTT​CAA​CAT​C.

Eight- to 10-week-old female Balb/c athymic nude mice (SPF Biotechnology, Beijing, China) were housed in standard facilities. Human U251/TMZ cells in PBS were injected intracranially, 2.0 mm below the skull surface, according to a previously published protocol (Ji et al., 2018). Three days after surgery, mice were randomized to receive one of the four following treatments: a drug vehicle (delivered orally once per day for 9 days). TMZ (50 mg/kg, delivered orally once per day for 5 days), or XH30 (5 mg/kg, delivered orally once per day for 9 days). TMZ and XH30 were dissolved in 0.5% carboxymethylcellulose solution. Tumor volumes were monitored using an animal magnetic resonance imaging (MRI) scanner (PharmaScan 70/16 US, Bruker, Germany). The parameters for the MRI scans were as follows: a T2_TurboRARE, with TR/TE = 5,000/40, 6 averages, 20 × 20 field of view, and 0.5-mm slice thickness. The tumor volume on the basis of MRI was calculated as V = L × W × T, where L is the maximum length of tumor, W is the maximum width perpendicular to L, and T is the thickness of the tumor slice (set at 0.5 mm).

For the subcutaneous mice tumor model, female Balb/c athymic nude mice (eight to 10 weeks of age) were subcutaneously implanted with 1 × 10⁷ U251/TMZ cells in 0.2 ml matrigel solution in the right flank. After 2 weeks, tumor issue was harvested sterilely, and tumor cells were extracted from the tissue homogenate. Then, the mice were implanted with 2 × 10⁶ tumor cells each in the right flank. Seven days later, when the average tumor volumes reached to 100–300 mm³, the mice were randomized into four groups, in which either alone treatment or combination was administered, respectively, using the same dose regime as in the orthotopic model. Tumor volume and body weight were monitored twice a week. Tumor volume was calculated as V = 1/2 × L × W², where L is the maximum length of tumor and W is the maximum width of tumor. The mice were euthanized at day 14, and tumor tissues were collected for immunoblotting.

All procedures were approved by the Ethics Committee for Animal Experiments of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College and conducted under the Guidelines for Animal Experiments of Peking Union Medical College.

Most statistical analyses were performed utilizing GraphPad Prism v8.0.1 (La Jolla, CA, United States), and significance levels were evaluated using analysis of variance (ANOVA) or T-tests, as appropriate. In our experiments, we distinguish between three of significance (***p < 0.001, **p < 0.01, and *p < 0.05, respectively).

On the basis of the cell viability assay, the combination index (CI) was calculated using the Chou–Talalay method, with CI = 1, CI < 1 and CI > 1 denoting an additive effect, synergism, and antagonism, respectively. CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (Dx)1 and (Dx)2 represented concentrations of each drug alone to exert x% effect, while (D)1 and (D)2 were concentrations of drugs in combination to elicit the same effect.

Here, we assessed the anti-tumor activity of XH30 in two TMZ-resistant GBM cell lines: one with acquired TMZ resistance and overactivated hedgehog signaling (U251/TMZ) and one with natural TMZ resistance and elevated MGMT levels (T98G). As shown in Figure 1A, TMZ did not exhibit cytotoxicity at the highest concentration of 1,000 μM. XH30 suppressed cell proliferation of both U251/TMZ and T98G cells, with 72 h IC₅₀ values of 191 and 183 nM, respectively. The cytotoxic effect was stronger at 72 h than at 24 or 48 h (Supplementary Figure S1). The positive control, PF-04691502, also inhibited the proliferation of both cell types. In a colony formation assay, the formation of cell colonies dose-dependently decreased after exposure to XH30 in both U251/TMZ and T98G cells (Figures 1B–E). These results indicated that XH30 exhibited strong inhibitory effects on the proliferation of TMZ-resistant GBM cells.

We assessed the inhibitory activity of XH30 on the PI3K signaling pathway in both U251/TMZ and T98G cells. XH30 dose-dependently blocked downstream molecules in the PI3K pathway including p-AKT, p-GSK3β, p-PRAS40, p-p70S6K, and p-S6RP (Figures 2A–D). At a concentration of 100 nM, XH30 strongly suppressed the phosphorylation of these signaling molecules. This inhibitory activity was more potent than the inhibitory effect of the positive control, PF-04691502, at the same concentrations.

Next, we investigated whether XH30 could induce cell cycle arrest or apoptosis in both TMZ-resistant GBM cell lines. In U251/TMZ cells, both PF-04691502 and XH30 induced cell cycle arrest in the G0/G1 phases. The percentage of G1 phase increased from 48.62% to 76.30% after a concentration titration of XH30 (Figure 3A). In T98MG cells, cell cycle arrest was observed during the G1 phase after exposure to XH30. The percentage of G1 phase at a XH30 concentration of 500 nM was increased compared to the control group (77.78% vs. 50.07%) (Figure 3B). Moreover, XH30 downregulated the expression of cyclin D1 and CDK2, which are markers of the G1 phase (Figures 3C,D). However, apoptosis was not observed in either U251/TMZ or T98G cells after exposure to XH30 for 48 h. These data demonstrated that XH30 induces cell cycle arrest in TMZ-resistant GBM cells (Figures 3E,F).

To further explore the antitumor activity of XH30 in TMZ-resistant GBM, we used a U251/TMZ orthotopic mouse model. Mice were given TMZ orally at doses of 50 mg/kg/day for 5 days or XH30 at either 5 mg/kg or 10 mg/kg daily for 9 days. In our MRI analysis, the images showed that XH30 suppressed tumor growth in brain (Figures 4A,B). At a dose of 10 mg/kg/day, tumor volume was significantly lower compared to the control group that received the drug vehicle (p < 0.01; Figure 4C; Supplementary Table S1). In this model, TMZ did not reduce tumor volume at a dose of 50 mg/kg/day with 6.2% tumor growth inhibition (TGI), whereas the TGI of TMZ at the same dose was 98.4% in a U251 (TMZ sensitive) orthotopic mice model (Supplementary Figures S3A, B). During the experiments, the body weight of mice in the XH30 group did not significantly decrease compared to that of the control group (Supplementary Figure S2A). Together, these data indicate that XH30 suppressed TMZ-resistant GBM growth in vivo.

Because XH30 showed excellent antitumor activity against TMZ-resistant GBM and TMZ resistance is a current barrier to effective treatment in GBM, we investigated whether XH30 could sensitize GBM to TMZ. To do this, XH30 and TMZ were administered together in GBM cells. XH30 at concentrations of 20 and 100 nM enhanced TMZ cytotoxicity in both U251/TMZ and T98G cells (Figure 5A). Calculation of the CI gave values of <1 for combined XH30 and TMZ in both cell types, indicating a synergic effect of these drugs (Supplementary Table S2). Immunoblotting results showed that the combination of XH30 and TMZ increased the level of the DNA damage marker λH2AX to a greater extent compared with TMZ or XH30 alone, indicating that XH30 increases TMZ cytotoxicity (Figure 5B).

We then evaluated whether combining XH30 and TMZ had synergistic antitumor effects in vivo. We employed a subcutaneous mouse model implanted with U251/TMZ cells. The groups that received XH30 or a combination of XH30 and TMZ both had significantly delayed tumor growth (Figure 5C). Treatment with either TMZ at 50 mg/kg/day or XH30 at 5 mg/kg/day suppressed tumor growth, with TGI values of 58.9% and 69.9%, respectively (Figures 5D,E). Combined TMZ and XH30 significantly suppressed tumor growth compared with the groups that received either TMZ or XH30 alone, with a TGI of 90.1%. Although TMZ exhibited antitumor activity, the effect was weaker than in the parent cell line U251 (TMZ sensitive) in the subcutaneous mouse model (58.9% vs. 94.9% TGI, Figure S3C and D). The body weight change with combined TMZ and XH30 treatment was within acceptable limits (Supplementary Figure S4). The immunoblotting data also showed that the phosphorylation proteins downstream to PI3K including mTOR, AKT, and S6RP decreased in tumor tissues with XH30 alone and in combination with TMZ (Figure 5F).

The hedgehog signaling pathway has been demonstrated to have a role in TMZ resistance (Li J. et al., 2016; Lee, 2016), and crosstalk between noncanonical hedgehog and PI3K signaling pathways has been documented (Riobo et al., 2006). Previously, we showed that the hedgehog pathway is overactive in both U251/TMZ and T98G cells (Ji et al., 2018). Therefore, we hypothesized that inhibition of PI3K may also inhibit the noncanonical hedgehog pathway to increase the response to TMZ. As shown in Figure 6A, the protein levels of GLI1, a key factor in the hedgehog signaling pathway, was dose-dependently decreased in U251/TMZ cells exposed to XH30 in various concentrations, along with decreased levels of phosphorylated AKT. SMO protein levels did not decrease after treatment with XH30. Similar results were also observed in T98G cells. GLI1 target genes, such as PAX6 and GLI1 itself, were downregulated in the presence of XH30 at 100 nM (Figure 6B), likely because XH30 suppresses noncanonical hedgehog signaling pathway via the blockade of PI3K. In T98G cells, another GLI1 target gene, MGMT, was also downregulated by XH30. Next, we investigated whether XH30 was able to block the hedgehog pathway in the presence of the hedgehog ligand SHH (sonic hedgehog). As shown in Figure 6C, SHH activated the hedgehog pathway in U251/TMZ and T98G, reflecting upregulated GLI1 expression, whereas XH30 reduced GLI1 expression. In the presence of SHH, XH30 partially reversed SHH-mediated GLI1 activation. The phosphorylation of downstream proteins in the PI3K pathway, including AKT and S6RP, was consistently decreased in cells exposed to XH30. Moreover, insulin-like growth factor 1 (IGF-1), which activates PI3K, also upregulated GLI1 expression, which can be attributed to the crosstalk between the PI3K signaling pathway and GLI1 (Figure 6D). In the presence of XH30, IGF-mediated elevation of GLI1 was partially attenuated. There was no obvious change in SMO protein levels. We also observed that XH30 and TMZ in combination maintained lower level of GLI1 expression in both mRNA and protein level in TMZ-resistant GBM cells (Figures 6E,F). Immunoblotting results of tumor tissues also showed that the protein levels of GLI1 decreased in XH30 and TMZ combination group compared to vehicle group (Figure 6G). Together, our results demonstrate that XH30 suppressed GLI1 via blockade of the noncanonical hedgehog signaling pathway.