BIX-01294 inhibits the proliferation of esophageal squamous cell carcinoma cells by inducing DNA damage and activating the mitochondrial apoptosis pathway_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

WU Zhongjie ^1,2 , ZHANG Yanfei ² , LV Wang ¹ , SHENG Hongxu ¹ , ZHU Linhai ¹ , HU Yi ² ,  HU Jian ¹

1. Department of Thoracic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, P.R.China;
2. Department of Thoracic Surgery, The First Hospital of Jiaxing, Jiaxing, 314001, Zhejiang, P.R.China;

Corresponding author：

HU Jian, Email: dr_hujian@zju.edu.cn

Keywords：

Esophageal squamous cell carcinoma; histone methylase G9a; BIX-01294; DNA double-strand break; mitochondrial apoptosis pathway

DOI：

10.7507/1007-4848.202009031

Video：

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Objective To explore the effects and molecular mechanisms of histone methylase G9a inhibitor BIX-01294 on apoptosis in esophageal squamous cell carcinoma (ESCC).Methods MTT assay and Colony-forming Units were adopted to determine the effects of BIX-01294 on the growth and proliferation of ESCC cell lines EC109 and KYSE150. Flow cytometry was used to analyze the apoptosis status of ESCC cells after the treatment of BIX-01294. The effects of BIX-01294 treatment on the expressions of G9a catalytic product H3K9me2, DNA double-strand break (DSB) markers, and apoptosis-related proteins were detected by Western blotting.Results BIX-01294 inhibited the growth of EC109 and KYSE150 cells in a dose-dependent manner (P<0.05), and BIX-01294 with the inhibitory concentration 50% (IC₅₀) significantly inhibited the formation of colony (P<0.05). After 24 hours treatment of BIX-01294 (IC₅₀), the apoptosis rate of EC109 cells increased from 11.5%±2.1% to 42.5%±5.4%, and KYSE150 cells from 7.5%±0.9% to 49.2%±5.2% (P<0.05). The expression level of the G9a catalytic product, H3K9me2, significantly decreased (P<0.05); while the expression of the DSB marker γH2AX was dramatically enhanced (P<0.05). We also found that the mitochondrial apoptosis pathway was activated and the expression levels of cleaved caspase3 and cleaved PARP were significantly elevated (P<0.05).Conclusion BIX-01294, the inhibitor of methyltransferase G9a, prompted apoptosis in ESCC cells by inducing DSB damage and activating mitochondrial apoptosis pathway.

Citation： WU Zhongjie, ZHANG Yanfei, LV Wang, SHENG Hongxu, ZHU Linhai, HU Yi, HU Jian. BIX-01294 inhibits the proliferation of esophageal squamous cell carcinoma cells by inducing DNA damage and activating the mitochondrial apoptosis pathway. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2021, 28(5): 571-577. doi: 10.7507/1007-4848.202009031 Copy

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8.	Zhang J, Wang Y, Shen Y, et al. G9a stimulates CRC growth by inducing p53 Lys373 dimethylation-dependent activation of Plk1. Theranostics, 2018, 8(10): 2884-2895.
9.	Huang T, Zhang P, Li W, et al. G9A promotes tumor cell growth and invasion by silencing CASP1 in non-small-cell lung cancer cells. Cell Death Dis, 2017, 8(4): e2726.
10.	Cha ST, Tan CT, Chang CC, et al. G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/ β-catenin pathway. Nat Cell Biol, 2016, 18(9): 993-1005.
11.	Zhang J, Yao D, Jiang Y, et al. Synthesis and biological evaluation of benzimidazole derivatives as the G9a Histone Methyltransferase inhibitors that induce autophagy and apoptosis of breast cancer cells. Bioorg Chem, 2017, 72: 168-181.
12.	Huang Y, Zou Y, Lin L, et al. Effect of BIX-01294 on proliferation, apoptosis and histone methylation of acute T lymphoblastic leukemia cells. Leuk Res, 2017, 62: 34-39.
13.	Yokoyama M, Chiba T, Zen Y, et al. Histone lysine methyltransferase G9a is a novel epigenetic target for the treatment of hepatocellular carcinoma. Oncotarget, 2017, 8(13): 21315-21326.
14.	Guo AS, Huang YQ, Ma XD, et al. Mechanism of G9a inhibitor BIX-01294 acting on U251 glioma cells. Mol Med Rep, 2016, 14(5): 4613-4621.
15.	Darzynkiewicz Z, Zhao H, Halicka HD, et al. DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis. Crit Rev Clin Lab Sci, 2012, 49(5-6): 199-217.
16.	Uzdensky A, Demyanenko S, Bibov M, et al. Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin. Tumour Biol, 2014, 35(8): 8225-8233.
17.	Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy. Nat Rev Mol Cell Biol, 2014, 15(1): 49-63.
18.	Kudelova J, Fleischmannova J, Adamova E, et al. Pharmacological caspase inhibitors: Research towards therapeutic perspectives. J Physiol Pharmacol, 2015, 66(4): 473-482.
19.	Li KC, Hua KT, Lin YS, et al. Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma. Mol Cancer, 2014, 13: 172.
20.	Ding J, Li T, Wang X, et al. The histone H3 methyltransferase G9A epigenetically activates the serine-glycine synthesis pathway to sustain cancer cell survival and proliferation. Cell Metab, 2013, 18(6): 896-907.

1. Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin, 2016, 66(2): 115-132.
2. Hsu PK, Chen HS, Liu CC, et al. Application of the eighth AJCC TNM staging system in patients with esophageal squamous cell carcinoma. Ann Thorac Surg, 2018, 105(5): 1516-1522.
3. Yen CY, Huang HW, Shu CW, et al. DNA methylation, histone acetylation and methylation of epigenetic modifications as a therapeutic approach for cancers. Cancer Lett, 2016, 373(2): 185-192.
4. Hyun K, Jeon J, Park K, et al. Writing, erasing and reading histone lysine methylations. Exp Mol Med, 2017, 49(4): e324.
5. Chen WL, Sun HP, Li DD, et al. G9a —an appealing antineoplastic target. Curr Cancer Drug Targets, 2017, 17(6): 555-568.
6. Zhang J, He P, Xi Y, et al. Down-regulation of G9a triggers DNA damage response and inhibits colorectal cancer cells proliferation. Oncotarget, 2015, 6(5): 2917-2927.
7. Casciello F, Windloch K, Gannon F, et al. Functional role of G9a histone methyltransferase in cancer. Front Immunol, 2015, 6: 487.
8. Zhang J, Wang Y, Shen Y, et al. G9a stimulates CRC growth by inducing p53 Lys373 dimethylation-dependent activation of Plk1. Theranostics, 2018, 8(10): 2884-2895.
9. Huang T, Zhang P, Li W, et al. G9A promotes tumor cell growth and invasion by silencing CASP1 in non-small-cell lung cancer cells. Cell Death Dis, 2017, 8(4): e2726.
10. Cha ST, Tan CT, Chang CC, et al. G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/ β-catenin pathway. Nat Cell Biol, 2016, 18(9): 993-1005.
11. Zhang J, Yao D, Jiang Y, et al. Synthesis and biological evaluation of benzimidazole derivatives as the G9a Histone Methyltransferase inhibitors that induce autophagy and apoptosis of breast cancer cells. Bioorg Chem, 2017, 72: 168-181.
12. Huang Y, Zou Y, Lin L, et al. Effect of BIX-01294 on proliferation, apoptosis and histone methylation of acute T lymphoblastic leukemia cells. Leuk Res, 2017, 62: 34-39.
13. Yokoyama M, Chiba T, Zen Y, et al. Histone lysine methyltransferase G9a is a novel epigenetic target for the treatment of hepatocellular carcinoma. Oncotarget, 2017, 8(13): 21315-21326.
14. Guo AS, Huang YQ, Ma XD, et al. Mechanism of G9a inhibitor BIX-01294 acting on U251 glioma cells. Mol Med Rep, 2016, 14(5): 4613-4621.
15. Darzynkiewicz Z, Zhao H, Halicka HD, et al. DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis. Crit Rev Clin Lab Sci, 2012, 49(5-6): 199-217.
16. Uzdensky A, Demyanenko S, Bibov M, et al. Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin. Tumour Biol, 2014, 35(8): 8225-8233.
17. Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy. Nat Rev Mol Cell Biol, 2014, 15(1): 49-63.
18. Kudelova J, Fleischmannova J, Adamova E, et al. Pharmacological caspase inhibitors: Research towards therapeutic perspectives. J Physiol Pharmacol, 2015, 66(4): 473-482.
19. Li KC, Hua KT, Lin YS, et al. Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma. Mol Cancer, 2014, 13: 172.
20. Ding J, Li T, Wang X, et al. The histone H3 methyltransferase G9A epigenetically activates the serine-glycine synthesis pathway to sustain cancer cell survival and proliferation. Cell Metab, 2013, 18(6): 896-907.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

BIX-01294 inhibits the proliferation of esophageal squamous cell carcinoma cells by inducing DNA damage and activating the mitochondrial apoptosis pathway

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