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ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes

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Abstract

This study aimed to investigate the potential role of pyruvate kinase M2 (PKM2) and extracellular regulated protein kinase (ERK) in arsenic-induced cell proliferation. L-02 cells were treated with 0.2 and 0.4 μmol/L As3+, glycolysis inhibitor (2-deoxy-D-glucose,2-DG), ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene, U0126] or transfected with PKM2 plasmid. Cell viability, proliferation, lactate acid production, and glucose intake capacity were determined by CCK-8 assay, EdU assay, lactic acid kit and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake kit, respectively. Also, levels of PKM2, phospho-PKM2S37, glucose transporter protein 1 (GLUT1), lactate dehydrogenase A (LDHA), ERK, and phospho-ERK were detected using Western blot and the subcellular localization of PKM2 in L-02 cells was detected by immunocytochemistry (ICC). Treatment with 0.2 and 0.4 μmol/L As3+ for 48 h increased the viability and proliferation of L-02 cells, the proportion of 2-NBDG+ cell and lactic acid in the culture medium, and GLUT1, LDHA, PKM2, phospho-PKM2S37, and phospho-ERK levels and PKM2 in nucleus. Compared with the 0.2 μmol/L As3+ treatment group, the lactic acid in the culture medium, cell proliferation and cell viability, and the expression of GLUT1 and LDHA were reduced in the group co-treated with siRNA-PKM2 and arsenic or in the group co-treated with U0126. Moreover, the arsenic-increased phospho-PKM2S37/PKM2 was decreased by U0126. Therefore, ERK/PKM2 plays a key role in the Warburg effect and proliferation of L-02 cells induced by arsenic, and also might be involved in arsenic-induced upregulation of GLUT1 and LDHA. This study provides a theoretical basis for further elucidating the carcinogenic mechanism of arsenic.

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Funding

This work was supported by the National Natural Science Foundation of China (81673109) and the National Natural Science Foundation of China (No. 81830099).

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Authors and Affiliations

  1. Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University, No.157 Baojian Road, Nangang District, Harbin, 150081, Heilongjiang Province, China

    Fanshuo Yin, Xin Zhang, Zaihong Zhang, Meichen Zhang, Yunyi Yin, Yanmei Yang & Yanhui Gao

  2. Key Lab of Etiology and Epidemiology, Education Bureau of Heilongjiang Province & Ministry of Health of P. R. China, Harbin Medical University, Harbin, 150081, Heilongjiang Province, China

    Fanshuo Yin, Xin Zhang, Zaihong Zhang, Meichen Zhang, Yunyi Yin, Yanmei Yang & Yanhui Gao

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  1. Fanshuo Yin
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  2. Xin Zhang
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  3. Zaihong Zhang
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All authors contributed to the study conception and design. All authors read and approved the final manuscript.

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Fig. S1:

2-DG antagonized low dose of arsenic-induced proliferation of L-02 cells. The effects of 0.2 μmol/L As3+ and/or 2-DG were detested using the Cell-Light EdU Apollo in vitro imaging kit. Data from three independent experiment are shown. *p<0.05 vs. control group. (PNG 728 kb)

High resolution image (TIF 2.98 mb)

Fig. S2:

U0126 inhibited the activation of ERK signaling pathway induced by low dose of arsenic. a The effects of treatment with 0.4 μmol/L As3+ on the level of phospho-ERK and ERK were detected using Western blot. Data from three independent experiment are shown; b The effects of 0.2 μmol/L As3+and/or U0126 were detected using Western blot. Data from three independent experiment are shown. *p<0.05 vs. control group. (PNG 368 kb)

High resolution image (TIF 1.18 mb)

Fig. S3:

U0126 antagonized low dose of arsenic-induced proliferation of L-02 cells. The effects of 0.2 μmol/L As3+ and/or U0126 were detested using the Cell-Light EdU Apollo in vitro imaging kit. Data from three independent experiment are shown. *p<0.05 vs. control group. (PNG 1139 kb)

High resolution image (TIF 121401 kb)

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Yin, F., Zhang, X., Zhang, Z. et al. ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes. Biol Trace Elem Res 202, 493–503 (2024). https://doi.org/10.1007/s12011-023-03706-z

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