Abstract
Autophagy plays a double-edged sword for cancer; particularly, mitophagy plays important roles in the selective degradation of damaged mitochondria. However, whether mitophagy is involved in killing effects of tumor cells by ionizing radiation (IR) and its underlying mechanism remain elusive. The purpose is to evaluate the effects of mitochondrial ROS (mROS) on autophagy after IR; furthermore, we hypothesized that KillerRed (KR) targeting mitochondria could induce mROS generation, subsequent mitochondrial depolarization, accumulation of Pink1, and recruitment of PARK2 to promote the mitophagy. Thereby, we would achieve a new strategy to enhance mROS accumulation and clarify the roles and mechanisms of radiosensitization by KR and IR. Our data demonstrated that IR might cause autophagy of both MCF-7 and HeLa cells, which is related to mitochondria and mROS, and the ROS scavenger N-acetylcysteine (NAC) could reduce the effects. Based on the theory, mitochondrial targeting vector sterile α- and HEAT/armadillo motif-containing protein 1- (Sarm1-) mtKR has been successfully constructed, and we found that ROS levels have significantly increased after light exposure. Furthermore, mitochondrial depolarization of HeLa cells was triggered, such as the decrease of Na+K+ ATPase, Ca2+Mg2+ ATPase, and mitochondrial respiratory complex I and III activities, and mitochondrial membrane potential (MMP) has significantly decreased, and voltage-dependent anion channel 1 (VDAC1) protein has significantly increased in the mitochondria. Additionally, HeLa cell proliferation was obviously inhibited, and the cell autophagic rates dramatically increased, which referred to the regulation of the Pink1/PARK2 pathway. These results indicated that mitophagy induced by mROS can initiate the sensitization of cancer cells to IR and might be regulated by the Pink1/PARK2 pathway.
Highlights
Radiotherapy remains the chief strategy for cancer therapy and draws great attention in the medical field
AntiCOX IV, anti-β-actin, and anti-GAPDH were purchased from Santa Cruz, CA, USA; anti-voltage-dependent anion channel 1 (VDAC1), anti-heat-shock protein 60 (HSP60), anti-protein kinase 1 (Pink1), and anti-PARK2 were purchased from Bioworld Technology, Inc., USA; and anti-microtubule-associated protein 1 light chain 3 (LC3), anti-p62, and anti-Tom20 were purchased from Cell Signaling Technology, Danvers, MA, USA
HeLa and MCF-7 cells were treated with NAC and ionizing radiation (IR) (8 Gy), and the autophagic rate was measured with MDC staining by Flow Cytometry (FCM)
Summary
Radiotherapy remains the chief strategy for cancer therapy and draws great attention in the medical field. To overcome the radioresistance or to enhance the radiosensitivity is important [3]. Some studies have showed that autophagy strengthens the anticancer effects of radiotherapy on patients with oral squamous cell carcinoma [4] and sensitizes cancer cells to radiotherapy [5]. Autophagy is a basic process of catabolism of cellular components, such as the cytosol, organelles, and protein aggregates. In the last few years, the biological importance and molecular mechanisms have been extensively studied [5]. Autophagosomes can contain virtually any cytoplasmic element, including cytosolic proteins and various membranous organelles, mitochondria, endoplasmic reticulum, and peroxisomes [6]. Autophagy contributes to maintain cellular homeostasis, which is made to be cytoprotective function; as a type II programmed cell death, autophagy
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