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Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway

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Tumor Biology

Abstract

Methyl methanesulfonate (MMS) is an alkylating agent that can induce cell death through apoptosis and necroptosis. The molecular mechanisms underlying MMS-induced apoptosis have been studied extensively; however, little is known about the mechanism for MMS-induced necroptosis. Therefore, we first established MMS-induced necroptosis model using human lung carcinoma A549 cells. It was found that, within a 24-h period, although MMS at concentrations of 50, 100, 200, 400, and 800 μM can induce DNA damage, only at higher concentrations (400 and 800 μM) MMS treatment lead to necroptosis in A549 cells, as it could be inhibited by the specific necroptotic inhibitor necrostatin-1, but not the specific apoptotic inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk). MMS-induced necroptosis was further confirmed by the induction of the necroptosis biomarkers including the depletion of cellular NADH and ATP and leakage of LDH. This necroptotic cell death was also concurrent with the increased expression of p53, p53-induced gene 3 (PIG-3), high mobility group box-1 protein (HMGB1), and receptor interaction protein kinase (RIP) but not the apoptosis-associated caspase-3 and caspase-9 proteins. Elevated reactive oxygen species (ROS) level was also involved in this process as the specific ROS inhibitor (4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC)) can inhibit the necroptotic cell death. Interestingly, knockdown of PIG-3 expression by small interfering RNA (siRNA) treatment can inhibit the generation of ROS. Taken together, these results suggest that MMS can induce necroptosis in A549 cells, probably through the PIG-3-ROS pathway.

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Abbreviations

MMS:

Methyl methanesulfonate

DSB:

Double-strand break

PIG-3:

p53-induced gene 3

H2DCF-DA:

Dichlorodihydrofluorescein diacetate

HMGB1:

High mobility group box-1 protein

RIP:

Receptor interaction protein kinase

APDC:

4-Amino-2,4-pyrrolidine-dicarboxylic acid

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Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (Nos. 81172692, 81373036, and 81202241); Zhejiang Provincial Department of Science and Technology (2013C14016); Ministry of Science and Technology, China (2009DFB30390); and Post Doctor Science Fundation of China (No. 2011M501356). J. Yang is a recipient of the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents.

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    Authors and Affiliations

    1. Suzhou Biological Technology Co. Ltd. of Centre Testing International Corporation, Kunshan, Jiangsu, 215300, China

      Ying Jiang

    2. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310003, China

      Ying Jiang & Shigang Shan

    3. Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang, 310058, China

      Linfeng Chi, Guanglin Zhang & Xinqiang Zhu

    4. Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang, 310058, China

      Xiangjing Gao

    5. Department of Toxicology, Hangzhou Normal University School of Medicine, Hangzhou, Zhejiang, 310016, China

      Hongjuan Li & Jun Yang

    6. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, 310003, China

      Jun Yang

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    Correspondence to Xinqiang Zhu or Jun Yang.

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    Ying Jiang and Shigang Shan contributed equally to this work.

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    Jiang, Y., Shan, S., Chi, L. et al. Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway. Tumor Biol. 37, 3785–3795 (2016). https://doi.org/10.1007/s13277-015-3531-y

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