Cell culture

U373-MG and U87-MG [20] cells, collected from the China Academia Sinica Cell Repository, were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing fetal bovine serum (FBS; 10%), glutamine (2 mmol/l), and penicillin and streptomycin (0.1 mg/ml each) in a 5% CO₂ atmosphere at 37°C. Cells were transfected using Lipofectamine 2000 (11668030, Thermo Fisher) as per the manufacturer’s instructions.

Reagents and drug preparation

Afatinib (Giotrif, Selleck Chemicals) was diluted with dimethyl sulfoxide (DMSO) to 10 mmol/l and stored at –20°C. For in vitro studies, afatinib was diluted with DMSO to various concentrations (1, 5, and 10 µM) and subsequently added to cells in DMEM without serum. For in vivo studies, afatinib was diluted with distilled water to 1.25 mg/ml.

PUMA knockdown shRNA plasmid was purchased from Santa Cruz Biotechnology. PUMA overexpression plasmid was purchased from Addgene. All siRNAs were synthesized by GenePharma. Control siRNA duplexes targeted the sequence 5ʹ-UUC UCC GAA CGU GUC ACG U-3ʹ. PUMA siRNA duplexes targeted the sequence of 5ʹ-CGG ACG ACC UCA ACG CAC A-3ʹ. Lipofectamine 2000 (Invitrogen) was used for transfection as per the manufacturer’s instructions.

Xenograft model in athymic mice and drug treatment

All animal experiment protocols were approved by our hospital. U373-MG cells and U373-MG cells (1 × 10⁷) with PUMA knockdown were subcutaneously injected to the right flanks of female nude mice, thereby creating tumor xenografts. U373-MG tumor-carrying mice were randomly classified into four groups (six mice per group) 5 days after tumor cell inoculation. Next, tumor size was determined and tumor volume (TV) was obtained using the following formula (the unit of parameters was “mm”): TV = 1/2 (length × width²) mm³. Optimal TV inhibition percentage (TVI%) was calculated as 100 – (TV treated/TV control × 100).

The mice were then subjected to afatinib or vehicle treatment (0.01 g/kg body weight) for 5 days per week, for 30 days (the start date was considered day 1). They were executed when in a particularly weak condition.

Western blotting (WB)

Cells were lysed using a RIPA lysis assay buffer (89901, Thermo Fisher), and the total amount of 100 µg of proteins were separated using 10% SDS–PAGE and then transferred onto Immobilon PVDF (0.45 µm) membranes, which were successively incubated with primary antibodies for 16 h and corresponding secondary antibodies for 60 min. The primary and secondary antibodies used in this study were as follows: Rabbit monoclonal anti-Bax antibody (1 : 2500, 2774S, CST), Mouse monoclonal anti-Bcl-2 antibody (1 : 2500, 15071S, CST), Rabbit polyclonal anti-PUMA antibody (1 : 1000, ab9643, Abcam), Goat anti-rabbit IgG H&L (HRP) (1 : 5000, ab205718, Abcam), and Goat anti-mouse IgG H&L (1 : 5000, ab6708, Abcam). The Mouse anti-beta Actin antibody (1 : 5000, ab8226, Abcam) and the Mouse anti-beta GAPDH antibody (1 : 5000, ab8245, Abcam) served as a loading control. The above incubation process was conducted at 4°C. Blots were visualized using chemiluminescence reagents (15159, Thermo Fisher) and a C-DiGit blot scanner.

Quantitative polymerase chain reaction (Q-PCR)

RNA was isolated with TRIzol reagent (15596026, Thermo Fisher), and electrophoresis of the products on 3.5% agarose gels was performed to validate the specific generation of the expected PCR product. Relative gene expression levels were determined with SYBR Green master mix using qRT-PCR with GAPDH, which is stably expressed among samples, as an internal reference. The PUMA and GAPDH were amplified using the following pairs of primers: PUMA forward 5ʹ-CTG CTG CCC GCT GCC TAC CT-3ʹ and PUMA reverse 5ʹ-CCG CTC GTA CTG TGC GTT GAG-3ʹ; GAPDH forward 5ʹ-AAT CCC ATC ACC ATC TTC C-3ʹ and GAPDH reverse 5ʹ-CAT CAC GCC ACA GTT TCC-3ʹ [21]. Reactions were conducted in triplicate in 0.02 ml of reaction solution in an ABI 7500 Real-Time PCR System. The amplification profile was denatured at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s, in which fluorescence was acquired. At the end of the PCR cycles, melting curve analyses were performed. ΔCt was calculated by subtracting the Ct values of GAPDH from the Ct values of the gene of interest. ΔΔCt was then calculated by subtracting ΔCt of the control from ΔCt of the sample. The 2^–ΔΔCT method was applied to calculate the copy number of target genes.

Cell proliferation and colony formation assay

Cell proliferation capacity was evaluated using the CCK-8 assay according to the manufacturer’s instructions. Cells were seeded into 96-well plates, CCK-8 (10 µl) was added to each well and cells were incubated for a further 2 h at 37°C. Optical density (OD) was measured at 450 nm using an auto-microplate reader (infinite M200, Tecan, Switzerland).

For the colony formation experiment, cells were cultured in 6-well plates and cultured for 7 days. They were then fixed in 4% formaldehyde for 20 min and stained with 1.0% crystal violet.

Annexin V-FITC/PI flow cytometry (FC)

Apoptosis was assessed using the Annexin V-FITC/PI FC (640914, BioLegend) test as previously described [22].

Statistical analysis

Data were expressed as mean ± SD. Significant differences among groups were assessed by SPSS software (12.0) using ANOVA and Tukey’s test, and between-group differences were analyzed using the t-test. Values of p < 0.05 indicated a significant difference.

Afatinib-induced inhibition of proliferation and growth of glioma cells

U373 and U87 cells were treated with afatinib at 1, 5, and 10 µM, based on a previous study in non-small cell lung carcinoma [23]. A Cell Counting Kit-8 (CCK-8) assay indicated that the proliferation rates of U373 and U87 cells significantly decreased owing to afatinib treatment at 36–72 h, depending on the afatinib concentration (Figures 1 A, B). The colony formation assay indicated that the growth of glioma cells declined after afatinib treatment in a dose-dependent manner (Figures 1 C, D). Altogether, these results indicate that afatinib repressed both the proliferation and growth rates of the glioma cells.

Figure 1

Glioma cell growth and proliferation suppressed by afatinib. U373 and U87 cells were exposed to afatinib of different concentrations (1, 5, and 10 μM). A, B – CCK-8 assay reveals that the proliferation rates of the two cell lines significantly decreased when treated with different concentrations of afatinib. C, D – Soft agar colony formation assay for glioma cells was used to detect the number of cells at 72 h after afatinib treatment. N = 3; *p < 0.05; **p < 0.01 (vs control group)

Effects of afatinib on cell apoptosis and related proteins

To explore how afatinib affects the apoptosis of glioma cells, U373 and U87 cells were treated with afatinib (10 µM) and then examined with Annexin V/PI FC. Afatinib treatment clearly promoted apoptosis in U373 and U87 cells (Figures 2 A, B). Next, apoptosis-related proteins, anti-apoptotic Bcl-2 and pro-apoptotic Bax, were examined using WB; it was demonstrated that afatinib upregulated the expression of Bax while downregulating Bcl-2 expression in both the cell lines (Figures 2 C, D).

Figure 2

Apoptosis of glioma cells regulated by afatinib. U373 and U87 cells were treated with afatinib (10 μM). A, B – Annexin V/PI staining results reveal that afatinib significantly increased the apoptosis rate in both glioma cell lines. The early apoptotic population of cells is indicated in the upper right quadrant of each plot. C, D – Western blotting results reveal that afatinib treatment regulated the expression of Bcl-2 and Bax in U373 and U87 cells. N = 3; **p < 0.01 (vs. control group)

Moreover, we examined the expression of key pro-apoptotic PUMA expression in both cell lines using afatinib treatment of different concentrations. Afatinib treatment appeared to elicit PUMA expression in a dose-dependent manner (Figures 3 A, B).

Figure 3

PUMA expression increased by afatinib. U373 and U87 cells were exposed to afatinib with different concentrations (1, 5, and 10 μM). A, B – Western blotting was used to show PUMA expression in U373 and U87 cells. N = 3

The role of PUMA in afatinib-induced mediation of cell growth and apoptosis

To investigate the effect of PUMA in the regulation of growth and apoptosis of glioma cells by afatinib, PUMA was deliberately overexpressed or silenced in the afatinib-treated U373 and U87 cells.

First, the overexpressing cells were investigated. In the PUMA-overexpressing U373 and U87 cells, there was an increase in the inhibition of cell growth caused by afatinib treatment (Figures 4 A, B). Annexin V/PI FC also indicated that PUMA overexpression led to an increase in the number of apoptotic cells induced by afatinib (Figures 4 C, D). Overexpression of PUMA also increased the expression of Bax and reduced the Bcl-2 level (Figures 4 E, F). PUMA was then silenced in afatinib-treated U373 and U87 cells by the transfection of siRNA-PUMA. The colony formation assay showed that silencing PUMA restored the colony number that had been impaired by afatinib in both cell lines (Figures 5 A, B). Silencing PUMA also reduced the number of apoptotic cells, which were elevated by afatinib treatment (Figures 5 C, D). Additionally, silencing reduced Bax expression and increased Bcl-2 expression in the afatinib-treated U373 and U87 cells (Figures 5 E, F). Altogether, these data indicate that in this study, in vitro afatinib treatment reduced glioma cell growth through PUMA-mediated apoptosis.

Figure 4

Influence of PUMA overexpression on growth and apoptosis of afatinib-treated glioma cells. U373 and U87 cells with overexpressed PUMA were subjected to 10 μM afatinib for 2 days. A, B – Soft agar colony formation assay was utilized to determine the cell number at 48 h after afatinib treatment. C, D – Annexin V/PI FC was employed to reveal the apoptosis rate in both glioma cell lines after treatment. The early apoptotic population of the cells is visible in the upper right quadrant of each plot. E, F – Western blotting results reveal that PUMA overexpression regulated the expression of Bcl-2 and Bax in U373 and U87 cells. N = 3; *p < 0.05 (vs. indicated group)

Figure 5

Influence of PUMA silencing on growth and apoptosis of afatinib-treated glioma cells. U373 and U87 cells with silenced PUMA were treated with 10 μM afatinib for 2 days. A, B – Soft agar colony formation assay was used to detect the number of cells at 48 h after afatinib treatment. C, D – Annexin V/PI FC was employed to reveal the apoptosis rate in both glioma cell lines after treatment. The early apoptotic population of the cells is visible in the upper right quadrant of each plot. E, F – Western blotting results reveal that PUMA silencing regulated the expression of Bcl-2 and Bax in U373 and U87 cells. N = 3; *p < 0.05 (vs. indicated group)

Reduction of xenograft tumors through PUMA by afatinib administration

To further reveal the effect of afatinib and PUMA on glioma xenograft tumors in nude mice, U373-MG cells or U373-MG cells with PUMA knockdown (KD) were injected into the right flanks of female nude mice. The successful KD of PUMA was confirmed by Q-PCR and WB (Figures 6 A, B). Afatinib administration clearly reduced the tumor weight (Figure 6 C), and afatinib-treated tumors grew at a significantly slower rate than the vehicle-treated tumors (Figure 6 D). In the KD tumors, the downregulation of PUMA notably reduced the anti-tumor efficiency of afatinib, as evidenced by a restored tumor weight and tumor growth rate (Figures 6 C, D). Additionally, WB data showed that in PUMA KD tumor cells, Bcl-2 expression was higher, whereas Bax expression was lower than those in WT tumor cells (Figure 6 B). Altogether, these data indicate that in vivo afatinib administration reduced xenograft tumors through PUMA.

Figure 6

Influence of afatinib administration on wild type (WT) and PUMA knockdown mouse xenograft tumors. U373-MG cells with PUMA knockdown (KD) or control cells were subcutaneously injected in nude mice (n = 6). A – Quantitative PCR revealed the mRNA expression of PUMA in WT and PUMA knockdown mouse xenograft tumors. B – Western blotting showed the protein expression of PUMA, Bcl-2, and Bax in WT and PUMA knockdown mouse xenograft tumors. C – Xenograft tumor weight was determined 25 days after injection. D – Tumor growth curve at 0–24 days after injection. N = 3; *p < 0.05l; **p < 0.01 (vs. control group)