Different mitochondrial fragmentation after irradiation with X-rays and carbon ions in HeLa cells and its influence on cellular apoptosis

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Abstract

Although mitochondria are known to play an important role in radiation-induced cellular damage, the mechanisms by which ionizing radiation modulates mitochondrial dynamics are largely unknown. In this study, human cervical carcinoma cell line HeLa was used to demonstrate the different modes of mitochondrial network in response to different quality radiations such as low linear energy transfer (LET) X-rays and high-LET carbon ions. Mitochondria fragmented into punctate and clustered ones upon carbon ion irradiation in a dose- and LET-dependent manner, which was associated with apoptotic cell death. In contrast, low-dose X-ray irradiation promoted mitochondrial fusion while mitochondrial fission was detected until the radiation dose was more than 1 Gy. This fission was driven by ERK1/2-mediated phosphorylation of Drp1 on Serine 616. Inhibition of mitochondrial fragmentation suppressed the radiation-induced apoptosis and thus enhanced the resistance of cells to carbon ions and high-dose X-rays, but not for cells irradiated with X-rays at the low dose. Our results suggest that radiations of different qualities cause diverse changes of mitochondrial dynamics in cancer cells, which play an important role in determining the cell fate.

Introduction

Radiotherapy is an essential modality of cancer therapy. It has been estimated that about 60% of all cancer diseases are cured by radiotherapy alone or in combination with surgery [1]. Previously, radiotherapy with photons (X- or γ-rays) may be the only option for tumor patients. However, for last two decades, the number of patients who received treatment with charged particles, such as protons and heavy ions (typically carbon ions), is rapidly increasing (http://www.ptcog.ch). Compared with radiotherapy with X-rays, charged particle therapy shows some unique properties in physics and/or biology [2]. One of the biological advantages of charged heavy particles like carbon ions is their higher relative biological effectiveness (RBE) in contrast with sparse ionizing radiation such as X-rays.

Generally, radiation-induced nuclear DNA damages have been regarded as the main cause of mutation and cell death [3]. However, mitochondrial damage elicited by radiation is also attracting more and more attentions [4]. The multiple functions of mitochondria allow them to sense cellular stress and contribute to cell adaptation to challenging micro-environment conditions, conferring a high degree of plasticity to tumor cells for growth and survival [5]. Mitochondria are highly dynamic organelles that change their morphology in response to cellular signaling and differentiation. Mitochondrial morphology is maintained by the balance between fusion and fission [6]. In mammals, mitochondrial fusion is regulated by two outer membrane (OM) GTPases such as mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), whereas the inner membrane (IM) GTPase optic atrophy 1 (OPA1) regulates fusion of the IM. The central players of mitochondria fission include the Dynamin-related protein 1 (Drp1), a GTPase, which is localized mainly in the cytoplasm and recruited to mitochondrial fission sites via interaction with OM receptor proteins, such as mitochondrial fission 1 protein (Fis1) [6,7]. A perturbation of this process is associated with mitochondrial dysfunction in various diseases, including aging [8], neurodegenerative diseases [6], diabetes [9] and tumor [10].

Pioneering work on the influence of radiation on mitochondrial dynamics has shown that γ-rays excited accelerated mitochondrial fission, which was coupled with delayed mitochondrial O·−2 production in normal human fibroblast-like cells [11]. Cytoplasmic irradiation using a precision microbeam resulted in mitochondrial fragmentation in human small airway epithelial cells [12]. Moreover, mitochondrial fission was also observed in adenocarcinoma cells after laser irradiation [13]. In contrast, live imaging of mitochondria in hippocampal neurons of Sprague-Dawley rats has revealed that mitochondrial fusion occurred 5 days after irradiation with X-rays of 0.2 Gy [14]. How ionizing radiations, especially high linear energy transfer (LET) carbon ions, influence mitochondrial dynamics remains unclear. Recently, our study has indicated that carbon ions could effectively induce mitochondrial fission and the level of mitochondrial fragmentation rendered either mitophagy or apoptosis as the response of mitochondrial damages to high-LET radiation in breast cancer cell lines MCF-7 and MDA-MB-231 [15]. In this study, different mitochondrial morphological characteristics in conjunction with the key factors of mitochondrial fission and fusion in human cervical carcinoma HeLa cell line after exposure to radiations of different qualities (X-rays and carbon ions, even carbon ions of different LET values) were further analyzed and the relationship between the morphological characteristics and mitochondrial damage responses to X-rays and carbon ions was examined.

Section snippets

Cell culture and reagents

Human cervical carcinoma cell line HeLa was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% (v/v) fetal bovine serum and kept at 37 °C, 5% CO2 in incubators. PD0325901 treatment: cells were treated with MEK inhibitor PD0325901 (200 nM, Selleck, S1036) for 1 h before irradiation. Its treatment was removed during irradiation and then

Changes of mitochondrial dynamics in response to different quality radiations

To determine whether the radiations of different qualities may change mitochondrial dynamics, HeLa cells were irradiated with X-rays or carbon ions of 70 keV/μm at various doses. In response to 0.2 Gy X-rays, the morphology of mitochondria in HeLa cells became longer/elongated compared to the control 4 h after irradiation, and the mean of the mitochondrial lengths increased from 9.7 μm to 13.1 μm, suggesting that mitochondrial fusion occurred. Cells exposed to X-rays of 0.5 Gy showed the same

Discussion

Although it has been mostly accepted that DNA is the key target of radiation, the potential contribution from cytoplasmic damages cannot be ignored. In this study, we systematically investigated the changes in mitochondrial dynamics and mitochondrial damage responses in HeLa cells exposed to low-LET X-rays and high-LET carbon ions.

Mitochondrial stress and dysfunction resulting from hypoxia, exposure to mitochondrial toxins, metabolic diseases and radiation affect the mitochondrial morphology [

Disclosure statement

No competing financial interests exist.

Funding

This work is jointly supported by the National Key Research Program of China (Grant No.2016YFC0904700, 2016YFC0904702), the NSFC-CAS Joint Fund for Research Based on Large-scaled Scientific Facilities (Grant No.U1532264), The National Natural Science Foundation of China (Grant No.11075191 and Grant No.11205217), National Natural Science Foundation of China Academy of Engineering Physics (Grant No.U1730133), The Natural Science Foundation of Gansu Province (Grant No.17JRSRA310).

Acknowledgements

The authors thank Dr. Ryoichi Hirayama for his helpful suggestions. They also thank the crews of HIRFL and HIMAC.

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