Regulation of temozolomide resistance in glioma cells via the RIP2/NF‐κB/MGMT pathway

Temozolomide (34219) was obtained from Sigma‐Aldrich. SC75741 (HY‐10496), SN50 (HY‐P0151), JSH‐23 (HY‐13982), and lomeguatrib (HY‐13668) were obtained from MedChemExpress. Primary antibodies against NF‐κB p65 (D14E12) (59674S, 1:1000), p‐NF‐κB p65 (Ser536) (93H1) (3033S, 1:1000), and IκBα (44D4) (4812, 1:1000) were purchased from Cell Signaling Technology. Primary antibodies against MGMT (ab69629) were purchased from Abcam. Primary antibodies against Lamin B1 (B‐10) (sc‐374015) were purchased from Santa Cruz Biotechnology. Primary antibodies against GAPDH (1A6) (MB001, 1:1000) were purchased from Bio‐World. Anti‐mouse IgG, HRP‐linked antibody, and anti‐rabbit IgG, HRP‐linked antibodies were purchased from Santa Cruz Biotechnology. Matrigel (354248) was obtained from BD Biosciences.

T98G, U87MG, and SW1783 glioma cell lines were purchased from the American Type Culture Collection (ATCC). The U251 glioma cell line was purchased from the Culture Collection of the Chinese Academy of Science. The medium for culturing SW1783 cells was RPMI 1640 (Invitrogen). The medium for culturing T98G, U87MG, and U251 cells was DMEM (high glucose) (Invitrogen). The four glioma cell lines were maintained in a 5% CO₂ atmosphere at 37°C in medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (HyClone), and 10% FBS. The resistant variants of the T98G/TR cells were developed by culturing initial T98G cells with increasing concentrations of TMZ (from 75 to 4800 μM). For U87MG/TR cells, the resistant variants were developed by culturing initial U87MG cells with increasing concentrations of TMZ (from 75 to 2400 μM). TMZ was added every 48 hours. Cell viability was analyzed every month using the CCK‐8 assay. IC50 values of T98G, U87MG, T98G/TR, and U87MG/TR are presented in Figure 1A. TMZ was added at a final concentration of 1 mM to maintain the resistant phenotypes till 1 week prior to the experiments.

Cell viability was evaluated using the CCK‐8 (Cell Counting Kit‐8; Solarbio) assay. The cell suspension (100 μl/well) was inoculated in a 96‐well plate, and the plate was pre‐incubated in a humidified incubator (37°C, 5% CO₂). The cells were treated for 72 hours. CCK‐8 solution (10 μl) was added to each well of the plate. The plate was incubated for 4 hours in the incubator. Absorbance was measured at 450 nm using an ELx800 plate reader (BioTek).

The plasmid pEX‐3 containing the RIP2 gene (NM_003821.1↗) was constructed by GenePharma, Inc., and transfection was performed using GenJet™ Plus DNA In vitro Transfection Reagent (Signagen, SL100499) in accordance with the manufacturer's protocol.

To inhibit RIP2 expression, small interfering RNA (siRNA) against RIP2 was used. RIP2‐targeting siRNA was designed and manufactured by GenePharma, Inc. The siRNA was added to 5 μl of RNase‐free water at a concentration of 100 pmol/μl, mixed with 10 μl of the transfection reagent, and maintained at 25°C for 15 minutes. For the transfection assay, 5 × 10⁶ cells were seeded in 6‐well plates and incubated overnight at 37°C in 5% CO₂ until 80%‐90% confluence was achieved and were then transfected with siRNA.

Cells were lysed with RIPA lysate to obtain total protein. Total proteins were separated by electrophoresis on 15% SDS‐PAGE and then transferred onto a PVDF membrane of 0.2 µm pore size (Millipore) for Western blot analysis. The PVDF membrane was blocked with 5% skim milk powder in Tris‐buffered saline/1% Tween‐20 (TBS‐T) for 2 hours at 4°C and then incubated with the primary antibodies anti‐RIP2 (monoclonal, rabbit anti‐human, 1:1000), anti‐NF‐κB p65 (monoclonal, rabbit anti‐human, 1:2000), anti‐p‐NF‐κB p65 (monoclonal, rabbit anti‐human, 1:8000), anti‐IκBα (monoclonal, mouse anti‐human, 1:1000), anti‐MGMT (monoclonal, rabbit anti‐human, 1:1500), anti‐LaminB (monoclonal, rabbit anti‐human, 1:1000), and anti‐GAPDH (monoclonal, rabbit anti‐human, 1:1000) (Cell Signaling Technology) overnight at 4°C. Afterward, the membranes were washed thrice with TBS‐T for 10 minutes and were incubated with horseradish peroxidase (HRP)‐labeled secondary antibodies of sheep to mouse and sheep to rabbit (ZSGB‐BIO Co., Ltd.) at 4°C for 1 hour. The non‐binding secondary antibodies were washed out thrice by TBS‐T for 10 minutes. Protein bands were detected using a SuperSignal West Femto Substrate (Thermo Fisher) and detected using a GE AI600 system (GE). Densitometric analysis of protein bands was performed using ImageQuant TL7.0 (GE).

A total of 5 × 10⁵ cells were collected and fixed in 70% ice cold ethanol overnight. Cells were then incubated with 10 mg/ml RNase (Sigma) and 50 mg/ml propidium iodide (Sigma) at 37°C for 30 minutes in dark. The treated cells were harvested and incubated with reagents from the Annexin V‐FITC apoptosis kit (BioVision) according to the manufacturer's protocol. Cell apoptosis was analyzed by flow cytometry (BD Bioscience).

Cells were plated on coverslips and then fixed with pre‐cooled methanol on ice for 20 minutes. Cells were then washed with PBST (PBS, 0.1% Triton X‐100), followed by blocking with 3% BSA at 15 ~ 25℃ for 30 minutes. Cells were incubated with primary antibodies for MGMT and then diluted in 3% BSA at 1:100 for 2 hours at room temperature. After washing thrice with PBST, the cells were incubated with secondary antibodies in 3% BSA for 2 hours. Nuclei were stained with 0.5 μg/ml of DAPI (PBST) at room temperature for 10 minutes. Cells were mounted with ProLong^® Gold Antifade (P36930↗, Thermo Fisher) and viewed with a confocal system under an FV1000 inverted microscope (Olympus). Images were analyzed using the scientific software module of Imaris.

Animal experiments were conducted in accordance with the Declaration of Helsinki and the Regulations for Care and Use of Laboratory Animals of the State Food and Drug Administration of China. The experimental protocols were approved by the Animal Ethics Committee of the Second Hospital of Hebei Medical University. Male nude mice (BALB/c‐nu) were obtained from GemPharmatech Co., Ltd. [License # SCXK(SU) 2018–0008] and maintained in a 12‐hour day/night cycle with free access to food and water. The glioma cells T98G/TR and U87MG/TR were resuspended in DMEM at 6 × 10⁶ cells per 200 µl and were injected subcutaneously into nude mice. Tumor diameter in the nude mice was measured daily using digital calipers. After the tumor volume reached 100 mm³, the mice were randomly divided into six groups (n = 6 mice per group): solvent control group, lomeguatrib group, JSH‐23 group, TMZ group, TMZ + lomeguatrib group, and TMZ + JSH‐23 group. TMZ (20 mg/kg i.p.), lomeguatrib (8 mg/kg i.p.), and JSH‐23 (3 mg/kg p.o.) were administered once a day for three weeks. The solvent control group was administered an equal volume of saline. Tumor sizes were measured thrice per week, and the volume was calculated using the formula: tumor volume (mm³) = (length × width²) × 1/2. The animals were anesthetized and sacrificed on day 21 after inoculation. Tumors were collected, weighed, and imaged and fixed with 10% neutral buffered formalin for pathological confirmation.

MGMT activity was quantified using the MGMT Activity Assay Kit (BioVision, Inc.) according to the manufacturer's instructions. Briefly, 2 × 10⁶ cells were resuspended in 100 μl extract buffer, put on ice for 20 minutes, and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatants were collected and diluted 10 times with ddH₂O, and the protein concentration was measured. The supernatants (containing ~50–200 μg of proteins) were diluted to 85 μl with extraction buffer. Positive and negative control wells were set up, and 10 μl of 10× reaction buffer and 5 μl calpain substrate were added into each well. After incubation in dark at 37°C for 1 hour, the fluorescence intensity of the samples was measured using a plate reader (BioTech) with excitation at 400 nm and emission at 505 nm.

Xenograft tumors were fixed with a 4% formaldehyde solution in PBS, embedded in paraffin, and sectioned. Following deparaffinization with xylene and hydration with decreasing concentrations of alcohol, the sections were incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity and boiled in EDTA buffer (pH = 8.0) for antigen retrieval. Sections were then incubated overnight with mouse monoclonal MGMT antibody at 4°C in a moist chamber. On the next day, after washing with PBS, the samples were incubated with HRP‐conjugated secondary antibody (ZSGB‐BIO Co., Ltd.) before microscopy analysis. The integrated optical density (IOD) values of tissue sections in each group were measured by Image‐Pro Plus 6.0 software (Media Cybernetics, Inc.).

All experiments were performed independently at least three times, and the data were analyzed using SPSS 19.0 and GraphPad Prism 7.0 for Windows. All data conform to the normal distribution by Shapiro‐Wilk test. All the results are expressed in terms of mean ± standard deviation (SD). Statistical significance was calculated using one‐way analysis of variance (ANOVA), followed by Fisher's multiple comparison test. p value <0.05 indicated statistical significance.

To explore the biological role of RIP2 in glioma cells, we first evaluated the viability of six types of glioma cells (T98G, T98G/TR, U87MG, U87MG/TR, U251, and SW1783) upon treatment with TMZ at different concentrations. Following TMZ treatment, T98G/TR and U87MG/TR cells had the highest viabilities and the highest TMZ inhibitory concentrations (IC50), followed by SW1783, whereas T98G, U87MG, and U251 cells had low viabilities and TMZ IC50 values, of which U251 cells were the least viable and had the lowest TMZ IC50 value (Figure 1A). Concurrently, we evaluated the differences in RIP2 expression in the six types of glioma cells. In each cell type, RIP2 expression shared positive correlation with cell viability and IC50 values (Figure 1B), suggesting an association between RIP2 expression and the effect of TMZ treatment in glioma cells.

To further confirm the role of RIP2 in TMZ chemoresistance, we induced RIP2 overexpression by transfecting the three types of glioma cells having low RIP2 expression with a RIP2 plasmid. Compared to that in the vector, the three types of cells transfected with RIP2 exhibited better viability upon TMZ treatment (Figure 1C‐G). Moreover, we used siRNA technology to hinder the expression of RIP2 in T98G/TR, U87MG/TR, and SW1783 cells. After RIP2 was disturbed, the viability of the three cells was significantly reduced (Figure 1F‐H). These results indicate that RIP2 is closely associated with resistance to TMZ in glioma cells.

Next, we silenced RIP2 expression in T98G/TR and U87MG/TR cells and subsequently, observed the sensitivity of the cells to TMZ to elucidate the underlying signaling mechanism. First, T98G/TR and U87MG/TR cells were treated with TMZ, stained with Annexin V‐FITC/PI, and observed using flow cytometry. Results indicated that after RIP2 silencing, total apoptosis of the two cell types increased significantly (Figure 2C). This implied that RIP2 silencing led to reduction in TMZ resistance in the two TMZ‐resistant cell lines. To further confirm the signaling mechanism, we detected NF‐κB signaling proteins using Western blotting. Analysis of the expression levels of NF‐κB p65, p‐NF‐κB p65, and IκBα in total cell proteins revealed that while RIP2 silencing did not affect the expression of total NF‐κB p65, it induced downregulation of p‐NF‐κB p65 expression and upregulation of IκBα expression (Figure 2A).

To confirm the regulatory mechanism of RIP2, we simultaneously monitored the response of RIP2‐overexpressing T98G and U87MG cells to TMZ treatment. Results of flow cytometry experiments indicated that RIP2 overexpression led to reduction in the total apoptosis rate of both cells (Figure 2D). RIP2 overexpression also led to reduced sensitivity to TMZ in T98G and U87MG cells. We observed that while RIP2 overexpression did not affect total NF‐κB p65 expression, it induced upregulation of p‐NF‐κB p65 expression and downregulation of IκBα expression in T98G and U87MG cells (Figure 2B). Analysis of protein expression in the nucleus revealed that NF‐κB p65 expression was significantly upregulated after RIP2 overexpression (Figure 2B). To conclude, RIP2 activates the NF‐κB signal pathway and reduces TMZ sensitivity in glioma cells.

O6‐methylguanine‐DNA methyltransferase (MGMT) is a DNA repair protein that repairs the damage inflicted by alkylating agents (such as TMZ) upon tumor cell DNA and thus imparts resistance to alkylating agents in tumor cells. Herein, we focused on the alterations in MGMT protein expression. MGMT expression was significantly downregulated in T98G/TR and U87MG/TR cells upon RIP2 silencing (Figure 3A), whereas it was significantly upregulated in T98G and U87MG cells upon RIP2 overexpression (Figure 3C). We further examined MGMT expression pattern in immunofluorescence experiments. The results were similar to those of Western blotting experiments (Figure 3B,D). RIP2 was observed to upregulate MGMT expression in glioma cells.

Our study revealed that RIP2 induces the NF‐κB signaling pathway and reduces the sensitivity of glioma cells to TMZ. RIP2 was observed to induce upregulation of MGMT expression in glioma cells. Whether RIP2 regulated MGMT expression by activating the NF‐κB pathway will need to be determined in further studies. In our study, we first used chemical inhibitors of NF‐κB (SC75741, SN50, and JSH‐23) to pretreat T98G/TR and U87MG/TR cells. After 48 hours, we extracted the total protein and observed that MGMT expression was downregulated in both cells (Figure 4A). Next, we studied RIP2 overexpression in T98G and U87MG cells. Results revealed that the three types of NF‐κB inhibitors inhibited RIP2 overexpression by varying protein expression (Figure 4B). Accordingly, we confirmed that RIP2 induces MGMT expression in glioma cells through the NF‐κB pathway.

We confirmed that RIP2 can regulate MGMT expression in glioma cells through the NF‐κB pathway. Considering that RIP2 can induce TMZ resistance in glioma cells, we pretreated the resistant cells (T98G/TR and U87MG/TR) and RIP2‐overexpressing cells (T98G and U87MG) with NF‐κB chemical inhibitors (SC75741, SN50, and JSH‐23) and an MGMT chemical inhibitor (lomeguatrib), respectively. After 48 hours of treatment with TMZ, TMZ sensitivity of the pretreated cells increased significantly (Figure 5A‐C). The results indicated that inhibition of NF‐κB or MGMT could enhance the sensitivity of glioma cells to TMZ.

We compared the expression of RIP2 in normal glioma xenograft tissues (T98G and U87MG) and TMZ‐resistant glioma xenograft tissues (T98G/TR and U87MG/TR). Results showed that RIP2 was upregulated in T98G/TR and U87MG/TR xenograft tissues (Figure 6A,B). To confirm the role of RIP2 in TMZ resistance in gliomas, we constructed the T98G/TR and U87MG/TR xenograft models. Immunohistochemical analysis revealed that the number of MGMT‐positive in cells pretreated with JSH‐23 reduced significantly, whereas lomeguatrib pretreatment did not affect MGMT expression (Figure 6C,D). Additionally, MGMT activity was significantly lower in the two xenografts pretreated with JSH‐23 or lomeguatrib than in the control group (Figure 6C,D). In the two xenograft tumor models, JSH‐23 or lomeguatrib itself has no obvious effect on tumor growth, but both can enhance the therapeutic effect of TMZ; thus, tumor volume is significantly reduced, and tumor weight is significantly reduced (Figure 7A,B).