Neuroprotective effect of oxytocin on cognitive dysfunction, DNA damage, and intracellular chloride disturbance in young mice after cranial irradiation.

Cranial radiation therapy (CRT) is an effective treatment for brain tumors; however, it also causes brain injuries. The pediatric brain is considered especially vulnerable compared to the adult brain; thus, brain injuries caused by CRT may severely affect their quality of life. In this study, we determined the neuroprotective effects of nasal oxytocin administration following cranial radiation in mice. We investigated the cognitive behavior of mice (novel object recognition test and novel object location test), phosphorylated histone H2AX (γ-H2AX) and K+-Cl- transporter (KCC2) by immunohistochemical analysis of the hippocampal sections, and neuronal cells by immunocytochemistry after radiation and oxytocin administration. We found that the number of γ-H2AX foci was increased, and the surface signal intensity of KCC2 immunofluorescence was decreased in cells that were irradiated with X-rays (1.5 Gy, for three consecutive days) compared with cells that were not. Furthermore, using MQAE, we found that the intracellular chloride ion concentration was downregulated in oxytocin-treated cells by increasing surface KCC2 expression. These results indicate that nasal oxytocin administration after cranial irradiation attenuates cognitive dysfunction in mice and exerts multifaceted neuroprotective effects on DNA damage and maintains chloride ion concentration in neuronal cells.

Dynamic imaging analysis reveals Auger electron-emitting radio-cisplatin induces DNA damage depending on the cell cycle.

Auger electrons can induce nanoscale physiochemical damage to DNA. The present study reports a sequential and systematic evaluation of the relationship between DNA damage such as double-strand breaks (DSBs) and the cell cycle for the Auger electron-emitting agent radiolabeled cisplatin with DNA binding ability. For dynamic imaging analysis, we used U2OS-derived cancer cells expressing two fluorescent fusion proteins: tumor-suppressor p53 binding protein 1 with a green fluorescent protein (53BP1-EGFP) and proliferating cell nuclear antigen with a red fluorescent protein (PCNA-DsRed). Time-lapse images of the cells were quantitatively analyzed using the ImageJ software with the deepImageJ plugin and the Google Colaboratory platform. From the middle-to-late G1 phase, around the G1-to-S phase transition, we found increased 53BP1 foci in cells treated with the radio-cisplatin. The radio-cisplatin caused significantly more DSBs than the nonradioactive cisplatin and saline in the G1 phase but not in the other phases. These results indicate that Auger electron-induced DNA damage, including DSBs, depends on the cell cycle. The G1 phase, which is associated with low DNA repair capacity and high radiosensitivity, is a promising target; thus, combining radiolabeled cisplatin with agents that arrest cells in the G1 phase could improve the DNA-damaging effect of Auger electrons and their therapeutic efficacy.

MiR-7-5p Is Involved in Ferroptosis Signaling and Radioresistance Thru the Generation of ROS in Radioresistant HeLa and SAS Cell Lines.

In cancer therapy, radioresistance or chemoresistance cells are major problems. We established clinically relevant radioresistant (CRR) cells that can survive over 30 days after 2 Gy/day X-ray exposures. These cells also show resistance to anticancer agents and hydrogen peroxide (H2O2). We have previously demonstrated that all the CRR cells examined had up-regulated miR-7-5p and after miR-7-5p knockdown, they lost radioresistance. However, the mechanism of losing radioresistance remains to be elucidated. Therefore, we investigated the role of miR-7-5p in radioresistance by knockdown of miR-7-5p using CRR cells. As a result, knockdown of miR-7-5p increased reactive oxygen species (ROS), mitochondrial membrane potential, and intracellular Fe2+ amount. Furthermore, miR-7-5p knockdown results in the down-regulation of the iron storage gene expression such as ferritin, up-regulation of the ferroptosis marker ALOX12 gene expression, and increases of Liperfluo amount. H2O2 treatment after ALOX12 overexpression led to the enhancement of intracellular H2O2 amount and lipid peroxidation. By contrast, miR-7-5p knockdown seemed not to be involved in COX-2 and glycolysis signaling but affected the morphology of CRR cells. These results indicate that miR-7-5p control radioresistance via ROS generation that leads to ferroptosis.

Lipid peroxidation increases hydrogen peroxide permeability leading to cell death in cancer cell lines that lack mtDNA.

4-Hydroxynonenal (HNE) is an important product of plasma membrane lipid peroxidation, which is a cause of cell and tissue injury. Mitochondrial DNA (mtDNA)-depleted ρ0 cells were established using human cervical cancer and oral squamous cell carcinoma cell lines. We investigated the effect of reactive oxygen species in ρ0 cells, especially the mechanism of hydrogen peroxide (H2 O2 )-mediated cell death. These cell were subjected to high oxidative stress and, compared with their parental cells, showed greater sensitivity to H2 O2 and high lipid peroxidation. Upregulation of HNE in the plasma membrane was observed prior to the increase in intracellular H2 O2 . The amount of oxidized lipid present changed H2 O2 permeability and administration of oxidized lipid led to further cell death after treatment with H2 O2 . Expression levels of lipoxygenase ALOX genes (ie ALOX5, ALOX12, and ALOX15) were upregulated in ρ0 cells, as were expression levels of ALOX12 and ALOX15 proteins. ALOX5 protein was mainly distributed in the nucleus, while ALOX12 and ALOX15 proteins were distributed in the nucleus and the cytoplasm. Although expression of COX2 gene was upregulated, its protein expression did not increase. ALOX (especially ALOX15) may be involved in the sensitivity of cancer cells to treatment. These data offer promise for the development of novel anticancer agents by altering the oxidation state of the plasma membrane. Our results showed that lipid peroxidation status is important for H2 O2 sensitivity and that ALOX15 is involved in lipid peroxidation status.

MiR-7-5p is a key factor that controls radioresistance via intracellular Fe2+ content in clinically relevant radioresistant cells.

MicroRNA (miRNA) is a non-coding RNA involved in regulating both cancer gene promotion and suppression. We investigated the role of miRNA in inducing radiation resistance in cancer cell lines using clinically relevant radioresistant (CRR) cells. Analysis using miRNA arrays and qPCR revealed that miR-7-5p is highly expressed in all examined CRR cells. Additionally, CRR cells lose their radioresistance when daily irradiation is interrupted for over 6 months. MiR-7-5p expression is reduced in these cells, and treating CRR cells with a miR-7-5p inhibitor leads to a loss of resistance to irradiation. Conversely, overexpression of miR-7-5p in CRR cells using a miR-7-5p mimic shows further resistance to radiation. Overexpression of miR-7-5p in parent cells also results in resistance to radiation. These results indicate that miR-7-5p may control radioresistance in various cancer cells at the clinically relevant dose of irradiation. Furthermore, miR-7-5p downregulates mitoferrin and reduces Fe2+, which influences ferroptosis. Our findings have great potential not only for examining radiation resistance prior to treatment but also for providing new therapeutic agents for treatment-resistant cancers.

Association between radiation-induced cell death and clinically relevant radioresistance.

Radiotherapy (RT) is one of the major modalities for the treatment of human cancer and has been established as an excellent local treatment for malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in RT. To know the characteristics of radioresistant cells, clinically relevant radioresistant (CRR) cell lines were established. CRR cells can continue to proliferate in vitro and in vivo after exposure to 2 Gy/day of X-rays for more than 30 days. Daily microscopic observation of the irradiated CRR cells has indicated that the increase in cell death is not observed within 7 days of irradiation with 10 Gy of X-rays, suggesting that cell death is involved in cellular radioresistance. Radiation-induced regulated cell death (RCD) can be classified into three categories: apoptosis, autophagy-dependent cell death and necrosis (necroptosis). This review focuses on an aspect of radiation-induced RCD that has often been neglected: the manner in which the cells are destroyed. In many studies, apoptosis is considered the primary mode of RCD in irradiated cancer cells; however, it is necessary to consider necrosis or necroptosis as one of the modes of radiation-induced RCD.

Clinically relevant radioresistant cells exhibit resistance to H2O2 by decreasing internal H2O2 and lipid peroxidation.

Radiation therapy is one of the choices to treat malignant tumors. In radiation therapy, existence of radiation-resistant cell is a major problem to overcome. We established clinically relevant radioresistant cells that had been obtained by exposing to 2 Gy/day X-rays for more than 30 days. These cells are resistant to 2 Gy/day X-ray exposure and anticancer agents. However, the underlying resistance mechanism remains unclear. We investigated the resistance of clinically relevant radioresistant cells to hydrogen peroxide (H2O2), confirming a degree of resistance. Neither catalase enzyme activity nor aquaporins appeared to be involved in H2O2 resistance. Mitochondrial DNA copy number, adenosine triphosphate (ATP) concentration, and plasma membrane potential were decreased. The timing of H2O2 intake was delayed and lipid peroxidation was decreased. Sensitivity of clinically relevant radioresistant cells to H2O2 was enhanced by 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine administration. These results suggest that the membrane status is a major factor conferring H2O2 resistance in clinically relevant radioresistant cells, and we should further investigate how membrane status could be used to enhance the therapeutic effect on cancer.

Sensitivity of mitochondrial DNA depleted ρ0 cells to H2O2 depends on the plasma membrane status.

To clarify the relationship between mitochondrial DNA (mtDNA)-depleted ρ0 cells and the cellular sensitivity to hydrogen peroxide (H2O2), we established HeLa and SAS ρ0 cell lines and investigated their survival rate in H2O2, radical scavenging enzymes, plasma membrane potential status, and chronological change in intracellular H2O2 amount under the existence of extracellular hydrogen peroxide compared with the parental cells. The results revealed that ρ0 cells had higher sensitivity to H2O2 than their parental cells, even though the catalase activity of ρ0 cells was up-regulated, and the membrane potential of the ρ0 cells was lower than their parental cells. Furthermore, the internal H2O2 amount significantly increased only in ρ0 cells after 50 µM H2O2 treatment for 1 h. These results suggest that plasma membrane status of ρ0 cells may cause degradation, and the change could lead to enhanced membrane permeability to H2O2. As a consequence, ρ0 cells have a higher H2O2 sensitivity than the parental cells.

Clinically relevant radioresistant cell line: a simple model to understand cancer radioresistance.

Radiotherapy (RT) is one of the major modalities for the treatment of human cancers and has been established as an excellent local treatment for malignant tumors. Conventional fractionated RT consists of 2-Gy X-rays, fractionated once a day, 5 days a week for 5-7 weeks in total 60 Gy. The efficacy of RT depends on the existence of radioresistant cells, which remains one of the most critical obstacles in RT and radio-chemotherapy. To improve the efficacy of RT, understanding the characteristics of radioresistant cells is one of the important subjects in radiation biology. Several studies have been reported to find out molecules implicated in radioresistance. However, it is noteworthy that cellular radioresistance has been mainly studied among cells with different genetic backgrounds and different origins. Therefore, making a system to compare between radioresistant and sensitive cells with the isogenic background is required. In this review, some aspects of cellular radioresistance mainly focusing on clinically relevant radioresistant (CRR) cell lines that can continue to proliferate even under exposure to 2-Gy X-rays, once a day, for more than 30 days, which is consistent with the conventional fractionated RT are discussed.

Methyl CpG-binding protein isoform MeCP2_e2 is dispensable for Rett syndrome phenotypes but essential for embryo viability and placenta development.

Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.

NK314 potentiates antitumor activity with adult T-cell leukemia-lymphoma cells by inhibition of dual targets on topoisomerase II{alpha} and DNA-dependent protein kinase.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease, incurable by standard chemotherapy. NK314, a new anticancer agent possessing inhibitory activity specific for topoisomerase IIα (Top2α), inhibited the growth of various ATL cell lines (50% inhibitory concentration: 23-70nM) with more potent activity than that of etoposide. In addition to the induction of DNA double-strand breaks by inhibition of Top2α, NK314 induced degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), resulting in impaired DNA double-strand break repair. The contribution of DNA-PK to inhibition of cell growth was affirmed by the following results: NK314 inhibited cell growth of M059J (a DNA-PKcs-deficient cell line) and M059K (a cell line with DNA-PKcs present) with the same potency, whereas etoposide exhibited weak inhibition of cell growth with M059K cells. A DNA-PK specific inhibitor, NU7026, enhanced inhibitory activity of etoposide on M059K as well as on ATL cells. These results suggest that NK314 is a dual inhibitor of Top2α and DNA-PK. Because ATL cells express a high amount of DNA-PKcs, NK314 as a dual molecular targeting anticancer agent is a potential therapeutic tool for treatment of ATL.

The reversibility of cancer radioresistance: a novel potential way to identify factors contributing to tumor radioresistance.

To understand the molecular mechanisms responsible for radioresistance in cancer cells, we previously established clinically relevant radioresistant (CRR) cell lines from several human cancer cell lines. These CRR cells proliferate even under exposure to 2 Gy/day of X-rays for more than 30 days, which is a standard protocol for tumor radiotherapy. CRR cells received 2 Gy/day of X-rays to maintain their radioresistance (maintenance irradiation; MI). Interestingly, CRR cells that did not receive MI for more than a year lost their radioresistance, indicating that radiation-induced radioresistance is reversible. We designated these CRR-NoIR cells. Karyotyping of the parental and CRR cells revealed that the chromosomal composition of CRR cells is quite different from that of the parental cells. However, CRR and CRR-NoIR cells were more similar compared with the parental cells because CRR cells repair X-ray-induced DNA damage with higher fidelity. To identify the factor(s) involved in tumor radioresistance, previously published studies including ours have compared radioresistant cells to parental cells. In this review, we conclude that a comparison between CRR and CRR-NoIR cells, rather than parental cells, is the best way to identify factors involved in tumor radioresistance.

Therapeutic potential for KCC2-targeted neurological diseases.

Patients with neurological diseases, such as schizophrenia, tend to show low K+-Cl- co-transporter 2 (KCC2) levels in the brain. The cause of these diseases has been associated with stress and neuroinflammation. However, since the pathogenesis of these diseases is not yet fully investigated, drug therapy is still limited to symptomatic therapy. Targeting KCC2, which is mainly expressed in the brain, seems to be an appropriate approach in the treatment of these diseases. In this review, we aimed to discuss about stress and inflammation, KCC2 and Gamma-aminobutyric acid (GABA) function, diseases which decrease the KCC2 levels in the brain, factors that regulate KCC2 activity, and the possibility to overcome neuronal dysfunction targeting KCC2. We also aimed to discuss the relationships between neurological diseases and LPS caused by Porphyromonas gingivalis (P. g), which is a type of oral bacterium. Clinical trials on oxytocin, sirtuin 1 (SIRT1) activator, and transient receptor potential cation channel subfamily V Member 1 activator have been conducted to develop effective treatment methods. We believe that KCC2 modulators that regulate mitochondria, such as oxytocin, glycogen synthase kinase 3ß (GSK3ß), and SIRT1, can be potential targets for neurological diseases.

SIRT2 down-regulation in HeLa can induce p53 accumulation via p38 MAPK activation-dependent p300 decrease, eventually leading to apoptosis.

We previously reported that sirtuin 2 (SIRT2), a mammalian member of the NAD+-dependent protein deacetylases, participates in mitotic regulation, specifically, in efficient mitotic cell death caused by the spindle checkpoint. Here, we describe a novel function of SIRT2 that is different from mitotic regulation. SIRT2 down-regulation using siRNA caused apoptosis in cancer cell lines such as HeLa cells, but not in normal cells. The apoptosis was caused by p53 accumulation, which is mediated by p38 MAPK activation-dependent degradation of p300 and the subsequent MDM2 degradation. Sirtuin inhibitors are emerging as antitumor drugs, and this function has been ascribed to the inhibition of SIRT1, the most well-characterized sirtuin that deacetylases p53 to promote cell survival and also binds to other proteins in response to genotoxic stress. This study suggests that SIRT2 can be a novel molecular target for cancer therapy and provides a molecular basis for the efficacy of SIRT2 for future cancer therapy.

Oxytocin ameliorates KCC2 decrease induced by oral bacteria-derived LPS that affect rat primary cultured cells and PC-12 cells.

Inflammation, especially neuroinflammation, which is caused by stress, leads to central nervous system (CNS) dysfunction. Because lipopolysaccharides (LPSs) cause neuroinflammation, we investigated the effect of LPSs to CNS. In PC-12 cells, LPSs derived from oral bacteria reduced the expression of KCC2, a Cl- transporter. LPS derived from P. gingivalis (P. g) administered to rat primary cultured cells also reduced the KCC2 expression. However, LPSs derived from E. coli did not reduce the KCC2 expression. LPS treatment activated TLR4, IL-1ß, and REST gene expressions, which led to KCC2 inactivation in PC-12 cells. The mechanism of KCC2 has been shown to play an important role in brain maturation, function (such as the GABA switch), and behavioral problems, we investigated the GABA function. We found that the GABA function was changed from inhibitory to excitatory by the LPS derived from P. g treatment. We demonstrated that the GSK3ß also involved in the KCC2 reduction by LPS treatment. We show that oxytocin rescued the reduction in KCC2 expression caused by LPSs by inhibiting GSK3ß signaling but vasopressin could not. Considered together, our results indicate that the LPSs from oral bacteria but not the LPS from E. coli increase the risk for brain disorders and oxytocin might be a candidate to overcome the abnormal behavior caused by brain disorders such as psychiatric disorders.

Development of Novel 191Pt-Labeled Hoechst33258: 191Pt Is More Suitable than 111In for Targeting DNA.

This study aims to establish new labeling methods for no-carrier-added radio-Pt (191Pt) and to evaluate the in vitro properties of 191Pt-labeled agents compared with those of agents labeled with the common emitter 111In. 191Pt was complexed with the DNA-targeting dye Hoechst33258 via diethylenetriaminepentaacetic acid (DTPA) or the sulfur-containing amino acid cysteine (Cys). The intranuclear fractions of 191Pt- and 111In-labeled Hoechst33258 were comparable, indicating that the labeling for 191Pt via DTPA or Cys and the labeling for 111In via DTPA worked equally well. 191Pt showed a DNA-binding/cellular uptake ratio of more than 1 order of magnitude greater than that of 111In. [191Pt]Pt-Hoechst33258 labeled via Cys showed a higher cellular uptake than that labeled via DTPA, resulting in a very high DNA-binding fraction of [191Pt]Pt-Cys-Hoechst33258 and extensive DNA damage. Our labeling methods of radio-Pt, especially via Cys, promote the development of radio-Pt-based agents for use in Auger electron therapy targeting DNA.

Disrupted plasma membrane localization of equilibrative nucleoside transporter 2 in the chemoresistance of human pancreatic cells to gemcitabine (dFdCyd).

Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.

c-Jun N-terminal kinase activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of retinoic acid receptor α protein in hepatic cells.

We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis.

Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor ß1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation.

OBJECTIVE AND DESIGN: To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs). MATERIAL: Using human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor ß1 (TGFß1). RESULTS: PPC suppressed ROS which are induced by TGFß1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels. CONCLUSION: These results suggest that ROS and Nox4 induced by TGFß1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.

Decreased mitochondrial membrane potential is an indicator of radioresistant cancer cells.

AIMS: To overcome radioresistant cancer cells, clinically relevant radioresistant (CRR) cells were established. To maintain their radioresistance, CRR cells were exposed 2 Gy/day of X-rays daily (maintenance irradiation: MI). To understand whether the radioresistance induced by X-rays was reversible or irreversible, the difference between CRR cells and those without MI for a year (CRR-NoIR cells) was investigated by the mitochondrial function as an index. MAIN METHODS: Radiation sensitivity was determined by modified high density survival assay. Mitochondrial membrane potential (Δψm) was determined by 5,5',6,6'-tetrachloro-1,1', tetraethylbenzimidazolocarbo-cyanine iodide (JC-1) staining. Rapid Glucose-Galactose assay was performed to determine the shift in their energy metabolism from aerobic glycolysis to oxidative phosphorylation in CRR cells. Involvement of prohibitin-1 (PHB1) in Δψm was evaluated by knockdown of PHB1 gene followed by real-time PCR. KEY FINDINGS: CRR cells that exhibited resistant to 2 Gy/day X-ray lost their radioresistance after more than one year of culture without MI for a year. In addition, CRR cells lost their radioresistance when the mitochondria were activated by galactose. Furthermore, Δψm were increased and PHB1 expression was down-regulated, in the process of losing their radioresistance. SIGNIFICANCE: Our finding reveled that tune regulation of mitochondrial function is implicated in radioresistance phenotype of cancer cells. Moreover, as our findings indicate, though further studies are required to clarify the precise mechanisms underlying cancer cell radioresistance, radioresistant cells induced by irradiation and cancer stem cells that are originally radioresistant should be considered separately, the radioresistance of CRR cells is reversible.