Cancer Res Treat.  2021 Apr;53(2):367-377. 10.4143/crt.2020.506.

Mutant IDH1 Enhances Temozolomide Sensitivity via Regulation of the ATM/CHK2 Pathway in Glioma

• Lin L
• Cai J
• Tan Z
• Meng X
• Li R
• Li Y
• Jiang C

• KMID: 2514919
• DOI: http://doi.org/10.4143/crt.2020.506

Abstract

Purpose
Isocitrate dehydrogenase 1 (IDH1) mutations are the most common genetic abnormalities in low-grade gliomas and secondary glioblastomas. Glioma patients with these mutations had better clinical outcomes. However, the effect of IDH1 mutation on drug sensitivity is still under debate.
Materials and Methods
IDH1-R132H mutant cells were established by lentivirus. IDH1-R132H protein expression was confirmed by western blot. The expression of ataxia telangiectasia mutated (ATM) signaling pathway and apoptosis-related proteins were detected by immunofluorescence and western blot. Temozolomide (TMZ) induced cell apoptosis was detected by flow cytometry. Tumor cell proliferation was detected by Cell Counting Kit-8. In vivo nude mice were used to confirm the in vitro roles of IDH1 mutation.
Results
We established glioma cell lines that expressed IDH1-R132H mutation stably. We found that TMZ inhibited glioma cells proliferation more significantly in IDH1 mutant cells compared to wild type. The IC₅₀ of TMZ in IDH1-R132H mutant group was less than half that of wild-type group (p < 0.01). TMZ significantly induced more DNA damage (quantification of γH2AX expression in IDH1 mutation vs. wild type, p < 0.05) and apoptosis (quantification of AnnexinV+propidium iodide–cells in IDH1 mutation versus wild type, p < 0.01) in IDH1 mutant gliomas compared to wild-type gliomas. The ATM-associated DNA repair signal was impaired in IDH1 mutant cells. Inhibiting the ATM/checkpoint kinase 2DNA repair pathway further sensitized IDH1 mutant glioma cells to chemotherapy. We found that IDH1 mutation significantly inhibited tumor growth in vivo (the tumor size was analyzed statistically, p < 0.05). Moreover, we confirmed that gliomas with IDH1 mutation were more sensitive to TMZ in vivo compared to wild type significantly and the results were consistent with the in vitro experiment.
Conclusion
These results provide evidence that combination of TMZ and ATM inhibitor enhances the antitumor effect in IDH1 mutant gliomas.

Keyword

Isocitrate dehydrogenase 1; Temozolomide; Glioma; Ataxia telangiectasia mutated

Figure

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  Fig. 1 Isocitrate dehydrogenase 1 (IDH1) mutation inhibits temozolomide (TMZ) treated glioma cells proliferation. (A) IDH1 mutant cells that stably expressing IDH1-R132H mutants in Ln229 and U87 cell lines. IDH1-R132H expression was confirmed by western blot. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B, C) IDH1 mutant or IDH1 wild-type cells were treated with or without TMZ for 0 to 96 hours. Cell proliferation was detected by Cell Counting Kit-8. (D, E) TMZ dose-response curves of Ln229 and U87 cells with IDH1 mutation for 96 hours. (F) Statistical analysis of IC50 in Ln229 and U87 glioma cells. **p < 0.01.

• 

  Fig. 2 Temozolomide (TMZ) induced more DNA damage in isocitrate dehydrogenase 1 (IDH1) mutation cells. (A, B) Immunofluorescence staining of γH2AX in glioma cells Ln229 and U87. Scale bars=100 μm. (C, D) Quantification of γH2AX is shown in A and B. *p < 0.05. (E, F) γH2AX expression in glioma cells Ln229 and U87 were detected by western blotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 3 Isocitrate dehydrogenase 1 (IDH1) mutant enhance temozolomide (TMZ) induced apoptosis. (A) Flow cytometry apoptosis analysis of IDH1 mutant and wild-type cells treated TMZ at different time points. (B) Statistical quantification of annexin V+propidium iodide (PI)− cells. (C) Statistical quantification of annexin V+PI+ cells. (D) Apoptosis-related protein Bax and Bcl-2 were detected by western blotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (E, F) Quantification of relative expression of Bax and Bcl-2. **p < 0.01.

• 

  Fig. 4 Isocitrate dehydrogenase 1 (IDH1) mutation inhibits ataxia telangiectasia mutated (ATM)/checkpoint kinase 2 (CHK2) signaling triggered by temozolomide (TMZ). (A, B) The expression of ATM signaling including ATM, CHK2, p53, and their phosphorylated proteins in glioma cells treated with or without TMZ were detected by Western blotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 5 Ataxia telangiectasia mutated (ATM) inhibitor enhanced the effect of temozolomide (TMZ) on tumor apoptosis. (A) Isocitrate dehydrogenase 1 (IDH1) mutation and wild-type gliomas were divided into four groups treated with negative control, ATM inhibitor, TMZ and ATM inhibitor+TMZ, after 72 hours, cell apoptosis was detected by flow cytometry. (B) Statistical quantification of annexin V+propidium iodide (PI)− cells. (C) IDH1 mutation and wild-type gliomas were treated with negative control, ATM inhibitor, TMZ and ATM inhibitor+TMZ, respectively. Cell proliferation was detected by Cell Counting Kit-8. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 6 The effect of isocitrate dehydrogenase 1 (IDH1) mutation on tumor in vivo. (A) The same number of IDH1 mutant and wild-type glioma cells were injected into the subcutaneous of 4–6 weeks nude mice, and the mice were killed and photographed in the fifth week. (B) The tumor size was detected and analyzed statistically. (C) The tumor weight was detected and analyzed statistically. (D) The tumor tissue was paraffin-embedded and stained with immunohistochemistry to detect the expression of Ki-67. Scale bars=25 μm. (E) The results of immunohistochemistry were scored and analyzed statistically. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 7 The effect of temozolomide (TMZ) on isocitrate dehydrogenase 1 (IDH1) mutant glioma in vivo. (A) IDH1 mutant and wild-type glioma cells were subcutaneously injected into 4–6 weeks BALB/c nude mice. After transplanted for 4 weeks 20 mg/kg TMZ was administered intraperitoneally into mice for consecutive 7 days. All nude mice were killed in the sixth week. (B) The tumor volume was monitored and analyzed. (C) The tumor weight was monitored and analyzed. (D) Primary glioma sections from mouse models were stained with Bax and Bcl-2 for immunohistochemistry (IHC) assay. Scale bars=25 μm. (E, F) The results of IHC were scored and analyzed statistically. All results are expressed as the mean±standard deviation; n=5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Reference

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