Opioid receptor antagonists reduce motivated wheel-running behavior in mice.

We hypothesized that opioid receptor antagonists would inhibit motivated behavior produced by a natural reward. To evaluate motivated responses to a natural reward, mice were given access to running wheels for 71.5 h in a multi-configuration testing apparatus. In addition to a running wheel activity, locomotor activity (outside of the wheel), food and water intake, and access to a food container were measured in the apparatus. Mice were also tested separately for novel-object exploration to investigate whether naloxone affects behavior unrelated to natural reward. In untreated mice wheel running increased from day 1 to day 3. The selective µ-opioid receptor antagonist ß-funaltrexamine (ß-FNA) (5 mg/kg) slightly decreased wheel running, but did not affect the increase in wheel running from day 1 to day 3. The non-selective opioid receptor antagonist naloxone produced a greater reduction in wheel running than ß-FNA and eliminated the increase in wheel running that occurred over time in the other groups. Analysis of food access, locomotor behavior, and behavior in the novel-object test suggested that the reduction in wheel running was selective for this highly reinforcing behavior. These results indicate that opioid receptor antagonism reduces responses to the natural rewarding effects of wheel running and that these effects involve multiple opioid receptors since the non-selective opioid receptor antagonist had greater effects than the selective µ-opioid receptor antagonist. It is possible that at the doses employed, other receptor systems than opioid receptors might be involved, at least in part, in the effect of naloxone and ß-FNA.

Kamishoyosan and Kamikihito protect against decreased KCC2 expression induced by the P. gingivalis lipopolysaccharide treatment in PC-12 cells and improve behavioral abnormalities in male mice.

Kamishoyosan (KSS) and Kamikihito (KKT) have been traditionally prescribed for neuropsychiatric symptoms in Japan. However, the molecular mechanism of its effect is not elucidated enough. On the other hand, it has been reported that lipopolysaccharide derived from Porphyromonas gingivalis (P. g LPS) is involved not only in periodontal disease but also in the systemic diseases such as psychiatric disorders via neuroinflammation. Here, we investigated the molecular mechanism of KSS and KKT treatment by LPS-induced neuropathy using PC-12 cells. When P. g LPS was administrated during the NGF treatment, the KCC2 expression was decreased in PC-12 cells. P. g LPS treatment also decreased the WNK and phospho SPAK (pSPAK) expression and enhanced GSK-3ß expression that negatively regulates WNK-SPAK signaling. Moreover, when KSS or KKT was administrated before P. g LPS treatment, the decrease of KCC2, WNK and pSPAK was rescued. KSS and KKT treatment also rescued the enhancement of GSK3ß expression by P. g LPS treatment. Furthermore, KSS, KKT and/or oxytocin could rescue behavioral abnormalities caused by P. g LPS treatment by animal experiments. These effects were not shown in the Goreisan treatment, which has been reported to act on the central nervous system. These results indicate that KSS and KKT are candidates for therapeutic agents for neural dysfunction.

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.

Comparative data on emotional (psychotic) aggressive biting behavior in mice of ddY strain measured by using two devices; Aggressive response meter and powerlab-compatible type aggressive response meter.

The Aggressive Response Meter (ARM) has been validated for measuring emotional (psychotic) aggression triggered by mental irritation in mice. In the present article, we newly developed a device, pARM (PowerLab-compatible type ARM). We collected on the aggressive biting behavior (ABB) intensity and ABB frequency of 20 male and female mice of ddY strain studied over a period of 6 days by using pARM and the former ARM. We calculated Pearson's correlation between the values of pARM and those of ARM. The accumulated data can be referred as a basis for demonstrating the consistence of pARM and the former ARM, and used in future research to augment the understanding of stress-induced emotional aggression in mice.

Human umbilical cord-derived mesenchymal stem cells-harvested mitochondrial transplantation improved motor function in TBI models through rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation.

Each year, traumatic brain injury (TBI) causes a high rate of mortality throughout the world and those who survive have lasting disabilities. Given that the brain is a particularly dynamic organ with a high energy consumption rate, the inefficiency of current TBI treatment options highlights the necessity of repairing damaged brain tissue at the cellular and molecular levels, which according to research is aggravated due to ATP deficiency and reactive oxygen species surplus. Taking into account that mitochondria contribute to generating energy and controlling cellular stress, mitochondrial transplantation as a new treatment approach has lately reduced complications in a number of diseases by supplying healthy and functional mitochondria to the damaged tissue. For this reason, in this study, we used this technique to transplant human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)-derived mitochondria as a suitable source for mitochondrial isolation into rat models of TBI to examine its therapeutic benefit and the results showed that the successful mitochondrial internalisation in the neuronal cells significantly reduced the number of brain cells undergoing apoptosis, alleviated astrogliosis and microglia activation, retained normal brain morphology and cytoarchitecture, and improved sensorimotor functions in a rat model of TBI. These data indicate that human umbilical cord-derived mesenchymal stem cells-isolated mitochondrial transplantation improves motor function in a rat model of TBI via rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation, maybe as a result of restoring the lost mitochondrial content.

Glycogen Synthase Kinase-3 Inhibitors Block Morphine-Induced Locomotor Activation, Straub Tail, and Depression of Rearing in Mice Via a Possible Central Action.

We investigated morphine-induced Straub's tail reaction (STR) in mice pretreated with or without glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763 and AR-A014418) by using a newly modified, infrared beam sensor-based automated apparatus. Mice treated with a single injection of morphine (30 mg/kg, i.p.) showed a significant STR with a plateau level at a time point of 20 min after morphine challenge. Pretreatment of mice with SB216763 (5 mg/kg, s.c.) or AR-A014418 (3 mg/kg, i.p.) significantly inhibited morphine-induced STR and attenuated the duration of STR in a dose-dependent fashion. In the striatum and the nucleus accumbens, expression of pGSK-3ßTyr216 but not GSK3ß or pGSK-3ßSer9 was largely but not significantly reduced after treatment with SB216763 (5 mg/kg, s.c.) in combination with/without morphine, indicating that the inhibitory effect of GSK-3 inhibitors on morphine-induced STR and hyperlocomotion might not depend on the direct blockade of GSK-3ß function. In constipated mice after morphine challenge (30 mg/kg), the effect of GSK-3 inhibitors on gastrointestinal transit was examined to reveal whether the action of GSK-3 inhibitors on morphine effects was central and/or peripheral. Pretreatment with SB216763 (5 mg/kg) did not block constipation in morphine-injected mice. The mechanism of action seems to be central but not peripheral, although the underlying subcellular mechanism of GSK-3 inhibitors is not clear. Our measurement system is a useful tool for investigating the excitatory effects of morphine in experimental animals.

Mitochondrial transfer in PC-3 cells fingerprinted in ferroptosis sensitivity: a brand new approach targeting cancer metabolism.

Despite recent therapeutic advancements, cancer remains one of the leading causes of death worldwide, with mitochondrial dysfunction being associated with cancer initiation and progression, along with chemotherapeutic resistance and ferroptotic cell death failure; however, the significance of mitochondria in various cancer types remains a matter of debate for the moment. The aim of this study is to ascertain the outcome of transferring healthy mitochondria into the aggressive and rapidly proliferating prostate cancer (PC-3) cells and afterwards evaluate the efficacy of combination therapy with or without the ferroptosis inducer erastin. In this sense, normal mitochondria were first isolated from human umbilical cord-derived mesenchymal stem cells, human umbilical vein endothelial cells, and human embryonic kidney cells and were later transferred into PC-3 cells and rhodamine 6G-treated PC-3 cells exhibiting mitochondrial dysfunction. Next, cell proliferation and sensitivity to cisplatin were measured using Cell Counting Kit-8 and the Malondialdehyde Assay Lipid Peroxidation Kit, respectively, along with ferroptotic damage. Transferring the healthy mitochondria into PC-3 cells was observed to increase cell proliferation and rescue the cisplatin-induced cell death, but not the erastin-induced ferroptosis, as in mitochondrial transfer effectively enhanced erastin-mediated ferroptosis in PC-3 cells. Hence, the introduction of healthy mitochondria into the highly aggressive and proliferating cancer cells would be deemed a brand new therapeutic strategy for a variety of cancers.

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.

Development of a sensitive double TaqMan Probe-based qPCR Angle-Degree method to detect mutation frequencies.

We designed a method to examine the mutation frequencies of the A3243G mutation of mitochondrial DNA (mtDNA) in patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. We performed a qPCR assay using the FAM and VIC TaqMan probes, which detect the 3243G (mutated) and 3243A (wild-type) sequences of mtDNA, respectively. The results obtained by "degree" in a series of differential mutation frequencies were used to plot a standard curve of the mutation frequency. The standard curve was then applied for qPCR assays of the desired samples. The standard deviation (%) of the samples calculated using the standard curve for the TaqMan probe was 2.4 ± 1.5%. This method could be used to examine mutation frequencies in the context of diabetes, aging, cancer, and neurodegenerative diseases.

Mitochondria: how eminent in ageing and neurodegenerative disorders?

Numerous factors are implicated in the onset and progression of ageing and neurodegenerative disorders, with defects in cell energy supply and free radicals regulation designated as being the main functions of mitochondria and highly accentuated in plentiful studies. Hence, analysing the role of mitochondria as one of the main factors implicated in these disorders could undoubtedly come in handy with respect to disease prevention and treatment. In this review, first, we will explore how mitochondria account for neurodegenerative disorders and ageing and later will draw the various pathways contributing to mitochondrial dysfunction in their distinct way. Also, we will discuss the deviation-countering mechanisms, particularly mitophagy, a subset of autophagy known as a much larger cellular defence mechanism and regulatory system, along with its potential therapeutic effects. Last but not least, we will be highlighting the mitochondrial transfer experiments with animal models of neurodegenerative disorders.

4-Methylumebelliferone Enhances Radiosensitizing Effects of Radioresistant Oral Squamous Cell Carcinoma Cells via Hyaluronan Synthase 3 Suppression.

Radioresistant (RR) cells are poor prognostic factors for tumor recurrence and metastasis after radiotherapy. The hyaluronan (HA) synthesis inhibitor, 4-methylumbelliferone (4-MU), shows anti-tumor and anti-metastatic effects through suppressing HA synthase (HAS) expression in various cancer cells. We previously reported that the administration of 4-MU with X-ray irradiation enhanced radiosensitization. However, an effective sensitizer for radioresistant (RR) cells is yet to be established, and it is unknown whether 4-MU exerts radiosensitizing effects on RR cells. We investigated the radiosensitizing effects of 4-MU in RR cell models. This study revealed that 4-MU enhanced intracellular oxidative stress and suppressed the expression of cluster-of-differentiation (CD)-44 and cancer stem cell (CSC)-like phenotypes. Interestingly, eliminating extracellular HA using HA-degrading enzymes did not cause radiosensitization, whereas HAS3 knockdown using siRNA showed similar effects as 4-MU treatment. These results suggest that 4-MU treatment enhances radiosensitization of RR cells through enhancing oxidative stress and suppressing the CSC-like phenotype. Furthermore, the radiosensitizing mechanisms of 4-MU may involve HAS3 or intracellular HA synthesized by HAS3.

Natural killer cell immunotherapy in glioblastoma.

Glioblastoma (GBM) is one of the most difficult cancers to treat because GBM has the high therapeutic resistance. Recently, immunotherapies for GBM have been used instead of conventional treatments. Among them, Natural killer (NK) cell-based immunotherapy has the potential to treat GBM due to its properties such as the absence of restriction by antigen-antibody reaction and deep penetration into the tumor microenvironment. Especially, genetically engineered NK cells, such as chimeric antigen receptor (CAR)-NK cells, dual antigen-targeting CAR NK cells, and adapter chimeric antigen receptor NK cells are considered to be an important tool for GBM immunotherapy. Therefore, this review describes the recent efforts of NK cell-based immunotherapy in GBM patients. We also describe key receptors expressing on NK cells such as killer cell immunoglobulin-like receptor, CD16, and natural killer group 2, member D (NKG2DL) receptor and discuss the function and importance of these molecules.

CRISPR/Cas9-mediated knockout of Lcn2 in human breast cancer cell line MDA-MB-231 ameliorates erastin-mediated ferroptosis and increases cisplatin vulnerability.

AIMS: Lipocalin 2 (Lcn2) is an antioxidant-related protein upregulated in various cellular stress conditions, especially cancer. In this study, we abrogated Lcn2 expression in MDA-MB-231 breast cancer cells using the CRISPR/Cas9 technology and evaluated its effect on cellular proliferation, migration, and ferroptotic cell death. MAIN METHODS: Validated human Lcn2 CRISPR/Cas9 knockout (KO) and homology-directed repair (HDR) plasmids were co-transfected into MDA-MB-231 breast cancer cells. Lcn2 gene knockout was confirmed at the transcriptional and protein levels using reverse transcription (RT)-PCR and enzyme-linked immunosorbent assay (ELISA). Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cytotoxicity assay was performed in the presence or absence of erastin, cisplatin (CDDP), and ferrostatin-1 using the CCK-8 method. Ferroptosis level was measured using the malondialdehyde assay lipid peroxidation kit. The migration capacity of the cells was also evaluated using the scratch assay. KEY FINDINGS: Targeting Lcn2 using CRISPR/Cas9 reduced cellular proliferation and migration capability, and elevated the vulnerability of MDA-MB-231 cells to cisplatin. Furthermore, Lcn2 expression loss effectively promoted erastin-mediated ferroptosis in MDA-MB-231 cells. SIGNIFICANCE: Inhibition of Lcn2 is a potentially useful strategy for sensitizing MDA-MB-231 tumor cells to ferroptotic cell death.

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.

Are Histamine H3 Antagonists the Definitive Treatment for Acute Methamphetamine Intoxication?

BACKGROUND: Methamphetamine (METH) is classified as a Schedule II stimulant drug under the United Nations Convention on Psychotropic Substances of 1971. METH and other amphetamine analogues (AMPHs) are powerful addictive drugs. Treatments are needed to treat the symptoms of METH addiction, chronic METH use, and acute METH overdose. No effective treatment for METH abuse has been established because alterations of brain functions under the excessive intake of abused drug intake are largely irreversible due in part to brain damage that occurs in the course of chronic METH use. OBJECTIVE: Modulation of brain histamine neurotransmission is involved in several neuropsychiatric disorders, including substance use disorders. This review discusses the possible mechanisms underlying the therapeutic effects of histamine H3 receptor antagonists on symptoms of methamphetamine abuse. CONCLUSION: Treatment of mice with centrally acting histamine H3 receptor antagonists increases hypothalamic histamine contents and reduces high-dose METH effects while potentiating lowdose effects via histamine H3 receptors that bind released histamine. On the basis of experimental evidence, it is hypothesized that histamine H3 receptors may be an effective target for the treatment METH use disorder or other adverse effects of chronic METH use.

Non-coding RNAs in ferroptotic cancer cell death pathway: meet the new masters.

Despite the recent advances in cancer therapy, cancer chemoresistance looms large along with radioresistance, a major challenge in dire need of thorough and minute investigation. Not long ago, cancer cells were reported to have proven refractory to the ferroptotic cell death, a newly discovered form of regulated cell death (RCD), conspicuous enough to draw attention from scholars in terms of targeting ferroptosis as a prospective therapeutic strategy. However, our knowledge concerning the underlying molecular mechanisms through which cancer cells gain immunity against ferroptosis is still in its infancy. Of late, the implication of non-coding RNAs (ncRNAs), including circular RNAs (circRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) in ferroptosis has been disclosed. Nevertheless, precisely explaining the molecular mechanisms behind the contribution of ncRNAs to cancer radio/chemotherapy resistance remains a challenge, requiring further clarification. In this review, we have presented the latest available information on the ways and means of regulating ferroptosis by ncRNAs. Moreover, we have provided important insights about targeting ncRNAs implicated in ferroptosis with the hope of opening up new horizons for overcoming cancer treatment modalities. Though a long path awaits until we make this ambitious dream come true, recent progress in gene therapy, including gene-editing technology will aid us to be optimistic that ncRNAs-based ferroptosis targeting would soon be on stream as a novel therapeutic strategy for treating cancer.

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.

Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability.

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (-) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

Pharmacological Targeting of Ferroptosis in Cancer Treatment.

Ferroptosis is a non-apoptotic mode of Regulated Cell Death (RCD) driven by excessive accumulation of toxic lipid peroxides and iron overload. Ferroptosis could be triggered by inhibiting the antioxidant defense system and accumulating iron-dependent Reactive Oxygen Species (ROS) that react with polyunsaturated fatty acids in abundance. Emerging evidence over the past few years has revealed that ferroptosis is of great potential in inhibiting growth and metastasis and overcoming tumor cell resistance. Thus, targeting this form of cell death could be perceived as a potentially burgeoning approach in cancer treatment. This review briefly presents the underlying mechanisms of ferroptosis and further aims to discuss various types of existing drugs and natural compounds that could be potentially repurposed for targeting ferroptosis in tumor cells. This, in turn, will provide critical perspectives on future studies concerning ferroptosis-based cancer therapy.

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.