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

Abstract Background Temozolomide (TMZ) is a first‐line chemotherapy drug for the treatment of malignant glioma and resistance to it poses a major challenge. Receptor‐interacting protein 2 (RIP2) is associated with the malignant character of cancer cells. However, it remains unclear whether RIP2 is involved in TMZ resistance in glioma. Methods RIP2 expression was inhibited in TMZ‐resistant glioma cells and normal glioma cells by using small interfering RNA (siRNA) against RIP2. Plasmid transfection method was used to overexpress RIP2. Cell counting kit‐8 assays were performed to evaluate cell viability. Western blotting or immunofluorescence was performed to determine RIP2, NF‐κB, and MGMT expression in cells. Flow cytometry was used to investigate cell apoptosis. TMZ‐resistant glioma xenograft models were established to evaluate the role of the RIP2/NF‐κB/MGMT signaling pathway in drug resistance. Results We observed that RIP2 expression was upregulated in TMZ‐resistant glioma cells, whereas silencing of RIP2 expression enhanced cellular sensitivity to TMZ. Similarly, upon the induction of RIP2 overexpression, glioma cells developed resistance to TMZ. The molecular mechanism underlying the process indicated that RIP2 can activate the NF‐κB signaling pathway and upregulate the expression of O6‐methylguanine‐DNA methyltransferase (MGMT), following which the glioma cells develop drug resistance. In the TMZ‐resistant glioma xenograft model, treatment with JSH‐23 (an NF‐κB inhibitor) and lomeguatrib (an MGMT inhibitor) could enhance the sensitivity of the transplanted tumor to TMZ. Conclusion We report that the RIP2/NF‐κB/MGMT signaling pathway is involved in the regulation of TMZ resistance. Interference with NF‐κB or MGMT activity could constitute a novel strategy for the treatment of RIP2‐positive TMZ‐resistant glioma.

rapid progression and have poor prognosis. [1][2][3] Currently, surgical treatment, radiotherapy, and chemotherapy are the major treatment methods for malignant gliomas. Novel therapies, such as molecular targeted therapy and immunotherapy, have also yielded positive clinical outcomes. 4,5 However, the majority of gliomas are invasive and difficult to treat, primarily because the boundary between the tumors and the surrounding brain tissue is unclear. 6 Alkylating agents are important antitumor drugs used in clinical practice. 7,8 Among them, those that induce the formation of O6-methylguanine pose a major threat to cells and can induce mutation and death. These agents can cross the blood-brain barrier and are nearly 100% bioavailable. 9 Temozolomide (TMZ) significantly improves the prognosis of patients with malignant glioma.
Compared with traditional chemotherapy drugs, TMZ causes fewer adverse reactions and is the best first-line drug for glioma treatment. [10][11][12] However, ever since TMZ has been used in clinical practice, there have been multiple reports stating that the rate of clinical efficacy of TMZ is less than 45%. Few of the patients treated with TMZ reported that though the short-term effects were encouraging, the long-term effects were not ideal. [13][14][15] This is because tumor cells develop primary or secondary resistance to TMZ. Resistance of glioma cells to TMZ is affected by several factors, such as tumor stem cells and their microenvironment, the stress response of tumor cells to chemotherapy drugs, the permeability of drugs in tumor tissues, and DNA damage and repair. [16][17][18][19] Receptor-interacting protein 2 (RIP2) belongs to the RIP family of proteins and is expressed in various tissues. RIP2 interacts with a variety of proteins, participates in multi-channel signal transduction, and executes the associated physiological functions. It is considered to form an important link between innate immunity, adaptive immunity, and inflammation. 20 RIP2 was shown to activate NF-κB, promoting anti-taxol-and ceramide-induced apoptosis in TNBC cells. 21 RIP2 was also suggested to be involved in drug resistance in triple-negative breast cancer. Dong et al 22 reported that the paired box protein 5 (Pax5) interacts with RIP2 to promote NF-κB activation and drug resistance in B-lymphoproliferative disorders. In our previous study, we showed that RIP2 expression increased in gliomas; additionally, findings from in vitro studies indicated that RIP2 could activate the NF-κB and p38 signaling pathways and subsequently influence the biological behavior of malignant gliomas. 23 However, it remained unclear whether RIP2 is involved in TMZ resistance in glioma. In this paper, we report that RIP2 expression is upregulated in TMZ-resistant neuroglioma cells and that RIP2 silencing enhances cellular sensitivity to TMZ.
The biological effects of RIP2 are mediated through activation of NF-κB for upregulation of MGMT expression. In the TMZ-resistant glioma cell xenograft model, inhibition of NF-κB and MGMT enhances the response of the transplanted tumor to TMZ. These findings help determine the mechanism of RIP2 resistance to TMZ in gliomas. NF-κB and MGMT may serve as valuable therapeutic targets in RIP2-positive gliomas. The four glioma cell lines were maintained in a 5% CO 2 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 assay
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% CO 2 ). 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).

F I G U R E 1
Receptor-interacting protein 2 (RIP2) decreased the sensitivity of glioma cells to TMZ. (A) Viability of glioma cells was determined using the CCK-8 method 72 hours after treatment with TMZ (75, 150, 300, 600, 1200, and 2400 μM), and the IC50 value was calculated according to the results of the CCK-8 assay. (B) RIP2 expression was observed using Western blotting. The results were normalized to GAPDH expression and expressed in terms of fold change in comparison with expression in T98G cells. (C-E) T98G, U87MG, and U251 cells were transfected with RIP2 plasmid and empty vector, and the activity of each cell was determined after 72 hours of treatment with TMZ (75, 150, 300, 600, 1200, and 2400 μM). (F-H) T98G/TR, U87MG/TR, and SW1783 cells were transfected with RIP2 siRNA and NC siRNA, and the activity of each cell was determined after 72 hours of treatment with TMZ (150, 300, 600, 1200, 2400, and 4800 μM). Results in (A)-(H) are expressed in terms of mean ± SD from three independent experiments

| siRNA interference
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 × 10 6 cells were seeded in 6-well plates and incubated overnight at 37°C in 5% CO 2 until 80%-90% confluence was achieved and were then transfected with siRNA.

| Cell apoptosis analysis by flow cytometry
A total of 5 × 10 5 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).

| Immunofluorescence analysis
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.

| MGMT activity assay
MGMT activity was quantified using the MGMT Activity Assay Kit (BioVision, Inc.) according to the manufacturer's instructions.
Briefly, 2 × 10 6 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 ddH 2 O, 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.

| Data and statistical analysis
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.

| RIP2 plays a role in glioma cell resistance to TMZ
To explore the biological role of RIP2 in glioma cells, we first evalu- 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.

| RIP2 reduces the sensitivity of glioma cells to TMZ by regulating the NF-κB pathway
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.

| Changes in MGMT expression induced by RIP2 in glioma cells are mediated through NF-κB
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.

| Inhibition of NF-κB/MGMT can enhance TMZ sensitivity in the drug-resistant glioma xenotransplantation model
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 F I G U R E 2 RIP2 reduces the sensitivity of glioma cells to TMZ by regulating the NF-κB pathway. (A) Representative Western blot bands of p-NF-κB p65, NF-κB p65, IκBα, and nuclear NF-κB p65 extracted from T98G/TR and U87MG/TR cells transfected with RIP2 siRNA or NC siRNA. The results were normalized to GAPDH (LaminB) expression and expressed as the fold change in comparison with expression in the blank. (B) Expression of p-NF-κB p65, NF-κB p65, IκBα, and nuclear NF-κB p65 protein was observed in T98G/TR and U87MG/TR cells transfected with RIP2 plasmid and empty vector. The results were normalized to GAPDH (LaminB) expression and expressed as the fold change in comparison with expression in the blank. (C) T98G/TR and U87MG/TR cells transfected with RIP2 siRNA or NC siRNA were treated with TMZ (2400 μM) for 24 hours, and next, apoptosis was measured by flow cytometry. (D) T98G and U87MG cells transfected with RIP2 plasmid or empty vector were treated with TMZ (600 μM) for 24 hours, and next, apoptosis was measured by flow cytometry.

| D ISCUSS I ON
Temozolomide is a first-line chemotherapeutic drug for the treat- interaction. 20 RIP2 plays a physiological or pathological role in processes related to immunity and inflammation. Studies conducted in recent years have revealed that RIP2 is related to the proliferation, migration, invasion, and metastasis of malignant tumors. [23][24][25] RIP2 has also been reported to activate NF-κB to promote chemotherapy resistance in triple-negative breast cancer cells. 21 RIP2 interacts with PAX5 to promote NF-κB activation and drug resistance in B-lymphoproliferative disorders. 22 However, there is no evidence to confirm the association between RIP2 and drug resistance in gliomas.
In this study, we first observed the expression of RIP2 in six glioma cell lines, including two TMZ-resistant cell lines, and assessed the sensitivity of the cells to TMZ. We observed that RIP2 expression level was the highest in TMZ-resistant cell lines and was closely related to TMZ resistance in the other four cell lines. Concurrently, similarly to that observed in earlier reports, upregulation of RIP2 expression led to activation of the NF-κB signaling pathway. [21][22][23] NF-κB is a nuclear transcription factor, named based on its specific binding with the enhancer κb sequence of κ light chain gene of B cell immunoglobulin. NF-κB has been shown to play a role in drug resistance in malignant cancers such as glioma, 26 breast cancer, 27 myeloma, 28 ovarian cancer, 29 and melanoma. 30 The efficacy of TMZ primarily depends on bioactive MTIC, which plays a cytotoxic role by alkylating O6 and N7 in guanine residues in DNA. 31  In conclusion, our study revealed that RIP2 was upregulated in TMZ-resistant glioma cells in correlation to drug resistance. The RIP2/NF-κB/MGMT signaling pathway plays a vital role in the regulation of TMZ resistance. Therefore, combined treatment with an NF-κB/MGMT inhibitor and TMZ enhances the therapeutic efficacy of the latter in RIP2-positive TMZ-resistant glioma. University for assisting our study.

CO N FLI C T O F I NTE R E S T
The authors do not have any possible conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the Supplementary Material of this article.