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.

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.

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.

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.

Transfer of healthy fibroblast-derived mitochondria to HeLa ρ0 and SAS ρ0 cells recovers the proliferation capabilities of these cancer cells under conventional culture medium, but increase their sensitivity to cisplatin-induced apoptotic death.

Mitochondrial dysfunction is known to contribute to cancer initiation, progression, and chemo-and radio-resistance. However, the precise role of mitochondria in cancer is controversial. Hence, here we tried to further clarify the role of mitochondria in cancer by transferring healthy mitochondria to cancer cells, and also to cells with depleted mitochondrial DNA (ρ0). Healthy mitochondria were isolated from WI-38 cells and were transferred to HeLa, SAS, HeLa ρ0, and SAS ρ0 cells. Then, cell proliferation was verified. In addition, the cells were treated by different concentrations of cisplatin and assessed for apoptosis induction and quantifying the mRNA expression of apoptosis-related genes. Results revealed that incubation of the HeLa, SAS and HeLa ρ0 cells with 5 µg/ml of the isolated mitochondria for 24 h significantly (p < 0.001) increased cell proliferation compared to non-treated controls. Interestingly, the mitochondria transfer rescued the ρ0 cells and made them capable of growing under conventional culture medium. However, the number of apoptotic cells was significantly higher in the HeLa ρ0 cells that received the mitochondria (HeLa-Fibro-Mit) compared to the HeLa ρ0. Furthermore, the expression level of BCL-2 anti-apoptotic gene was down-regulated in both HeLa-Fibro-Mit and SAS-Fibro-Mit cell lines while the expression levels of the BAX, caspase8, caspase9, and AIF pro-apoptotic genes were upregulated. Our findings indicated that although the response of cancer cells to the mitochondria transfer is cancer-type dependent, but the introduction of normal exogenous mitochondria to some cancer cells might be considered as a potential novel therapeutic strategy.

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.

Mucosal IgM Antibody with d-Mannose Affinity in Fugu Takifugu rubripes Is Utilized by a Monogenean Parasite Heterobothrium okamotoi for Host Recognition.

How parasites recognize their definitive hosts is a mystery; however, parasitism is reportedly initiated by recognition of certain molecules on host surfaces. Fish ectoparasites make initial contact with their hosts at body surfaces, such as skin and gills, which are covered with mucosa that are similar to those of mammalian guts. Fish are among the most primitive vertebrates with immune systems that are equivalent to those in mammals, and they produce and secrete IgM into mucus. In this study, we showed that the monogenean parasite Heterobothrium okamotoi utilizes IgM to recognize its host, fugu Takifugu rubripes Oncomiracidia are infective larvae of H. okamotoi that shed their cilia and metamorphose into juveniles when exposed to purified d-mannose-binding fractions from fugu mucus. Using liquid chromatography-tandem mass spectrometry analysis, proteins contained in the fraction were identified as d-mannose-specific IgM with two d-mannose-binding lectins. However, although deciliation was significantly induced by IgM and was inhibited by d-mannose or a specific Ab against fugu IgM, other lectins had no effect, and IgM without d-mannose affinity induced deciliation to a limited degree. Subsequent immunofluorescent staining experiments showed that fugu d-mannose-specific IgM binds ciliated epidermal cells of oncomiracidium. These observations suggest that deciliation is triggered by binding of fugu IgM to cell surface Ags via Ag binding sites. Moreover, concentrations of d-mannose-binding IgM in gill mucus were sufficient to induce deciliation in vitro, indicating that H. okamotoi parasites initially use host Abs to colonize host gills.

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.

Retinal regeneration after injury induced by gamma-ray irradiation during early embryogenesis in medaka, Oryzias latipes.

PURPOSE: Zebrafish, a small fish model, exhibits a multipotent ability for retinal regeneration after damage throughout its lifetime. Compared with zebrafish, birds and mammals exhibit such a regenerative capacity only during the embryonic period, and this capacity decreases with age. In medaka, another small fish model that has also been used extensively in biological research, the retina's inner nuclear layer (INL) failed to regenerate after injury in the hatchling at eight days postfertilization (dpf). We characterized the regenerative process of the embryonic retina when the retinal injury occurred during the early embryonic period in medaka. METHODS: We employed a 10 Gy dose of gamma-ray irradiation to initiate retinal injury in medaka embryos at 3 dpf and performed histopathological analyses up to 21 dpf. RESULTS: One day after irradiation, numerous apoptotic neurons were observed in the INL; however, these neurons were rarely observed in the ciliary marginal zone and the photoreceptor layer. Numerous pyknotic cells were clustered in the irradiated retina until two days after irradiation. These disappeared four days after irradiation, but the abnormal bridging structures between the INL and ganglion cell layer (GCL) were present until 11 days after irradiation, and the neural layers were completely regenerated 18 days after irradiation. After gamma-ray irradiation, the spindle-like Müller glial cells in the INL became rounder but did not lose their ability to express SOX2. CONCLUSIONS: Irradiated retina at 3 dpf of medaka embryos could be completely regenerated at 18 days after irradiation (21 dpf), although the abnormal layer structures bridging the INL and GCL were transiently formed in the retinas of all the irradiated embryos. Four days after irradiation, embryonic medaka Müller glia were reduced in number but maintained SOX2 expression as in nonirradiated embryos. This finding contrasts with previous reports that 8 dpf medaka larvae could not fully regenerate damaged retinas because of loss of SOX2 expression.

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.

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.

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.

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.

Acquirement of the autonomic nervous system modulation evaluated by heart rate variability in medaka (Oryzias latipes).

Small teleosts have recently been established as models of human diseases. However, measuring heart rate by electrocardiography is highly invasive for small fish and not widely used. The physiological nature and function of vertebrate autonomic nervous system (ANS) modulation of the heart has traditionally been investigated in larvae, transparent but with an immature ANS, or in anesthetized adults, whose ANS activity may possibly be disturbed under anesthesia. Here, we defined the frequency characteristics of heart rate variability (HRV) modulated by the ANS from observations of heart movement in high-speed movie images and changes in ANS regulation under environmental stimulation in unanesthetized adult medaka (Oryzias latipes). The HRV was significantly reduced by atropine (1 mM) in the 0.25-0.65 Hz and by propranolol (100 µM) at 0.65-1.25 Hz range, suggesting that HRV in adult medaka is modulated by both the parasympathetic and sympathetic nervous systems within these frequency ranges. Such modulations of HRV by the ANS in adult medaka were remarkably suppressed under anesthesia and continuous exposure to light suppressed HRV only in the 0.25-0.65 Hz range, indicating parasympathetic withdrawal. Furthermore, pre-hatching embryos did not show HRV and the power of HRV developed as fish grew. These results strongly suggest that ANS modulation of the heart in adult medaka is frequency-dependent phenomenon, and that the impact of long-term environmental stimuli on ANS activities, in addition to development of ANS activities, can be precisely evaluated in medaka using the presented method.

Kamishoyosan (a Japanese traditional herbal formula), which effectively reduces the aggressive biting behavior of male and female mice, and potential regulation through increase of Tph1, Tph2, and Esr2 mRNA levels.

Kamishoyosan (KSS), a Japanese traditional herbal formula, is used to treat symptoms related to the autonomic nervous system in men and women; it is especially known for improving the symptoms of irritability (e.g., bad temper and persistent anger). Although clinical and ethological studies of KSS have been conducted, its efficacy in reducing irritability remains to be validated. In the present study, male and female ddY-strain mice were isolation-reared for 8 weeks (from the third postnatal week) to induce pathologically aggressive biting behavior (ABB), which was used as an indicator of irritability. The ABB of mice toward metal rods was measured using the Aggressive Response Meter. An intraperitoneal administration of KSS (100 mg/kg) effectively reduced ABB in male and female mice at 2 h after the administration; however, this effect was canceled by prior administration of WAY-100635 [a 5-hydroxytryptoamine (5-HT)-1A receptor antagonist; 0.5 mg/kg] and bicuculline (a type-A gamma-aminobutyric acid receptor antagonist; 1.0 mg/kg). Additionally, tamoxifen, ICI-182780, and G-15 (all estrogen receptor antagonists) inhibited the action of KSS in a dose-dependent manner. Furthermore, gene expression of tryptophan hydroxylase (Tph) 1 and Tph2 were increased and 5-HT immunofluorescence was slightly increased in the dorsal raphe nucleus (DRN) of isolation-reared mice administered with KSS. Collectively, these results indicate that KSS effectively reduces ABB in isolation-reared male and female mice through stimulation of 5-HT production in the DRN. Our findings also suggest that gene expression of estrogen receptor (Esr) 2 increased in the DRN might be associated with the reduction of ABB.

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.

Mitochondrial Dysfunction in Diseases, Longevity, and Treatment Resistance: Tuning Mitochondria Function as a Therapeutic Strategy.

Mitochondria are very important intracellular organelles because they have various functions. They produce ATP, are involved in cell signaling and cell death, and are a major source of reactive oxygen species (ROS). Mitochondria have their own DNA (mtDNA) and mutation of mtDNA or change the mtDNA copy numbers leads to disease, cancer chemo/radioresistance and aging including longevity. In this review, we discuss the mtDNA mutation, mitochondrial disease, longevity, and importance of mitochondrial dysfunction in cancer first. In the later part, we particularly focus on the role in cancer resistance and the mitochondrial condition such as mtDNA copy number, mitochondrial membrane potential, ROS levels, and ATP production. We suggest a therapeutic strategy employing mitochondrial transplantation (mtTP) for treatment-resistant cancer.

Strain difference in transgene-induced tumorigenesis and suppressive effect of ionizing radiation.

Transgenic expression in medaka of the Xiphophorus oncogene xmrk, under a pigment cell specific mitf promoter, induces hyperpigmentation and pigment cell tumors. In this study, we crossed the Hd-rR and HNI inbred strains because complete genome information is readily available for molecular and genetic analysis. We prepared an Hd-rR (p53+/-, p53-/-) and Hd-rR HNI hybrid (p53+/-) fish-based xmrk model system to study the progression of pigment cells from hyperpigmentation to malignant tumors on different genetic backgrounds. In all strains examined, most of the initial hyperpigmentation occurred in the posterior region. On the Hd-rR background, mitf:xmrk-induced tumorigenesis was less frequent in p53+/- fish than in p53-/- fish. The incidence of hyperpigmentation was more frequent in Hd-rR/HNI hybrids than in Hd-rR homozygotes; however, the frequency of malignant tumors was low, which suggested the presence of a tumor suppressor in HNI genetic background fish. The effects on tumorigenesis in xmrk-transgenic immature medaka of a single 1.3 Gy irradiation was assessed by quantifying tumor progression over 4 consecutive months. The results demonstrate that irradiation has a different level of suppressive effect on the frequency of hyperpigmentation in purebred Hd-rR compared with hybrids.