Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

This corrects the article DOI: 10.1038/onc.2014.355.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

Manganese superoxide dismutase (MnSOD) is a mitochondrially localized primary antioxidant enzyme, known to be essential for the survival of aerobic life and to have important roles in tumorigenesis. Here, we show that MnSOD deficiency in skin tissues of MnSOD-heterozygous knockout (Sod2(+/-)) mice leads to increased expresson of uncoupling proteins (UCPs). When MnSOD is deficient, superoxide radical and its resulting reactive oxygen species (ROS) activate ligand binding to peroxisome proliferator-activated receptor alpha (PPARα), suggesting that the activation of PPARα signaling is a major mechanism underlying MnSOD-dependent UCPs expression that consequently triggers the PI3K/Akt/mTOR pathway, leading to increased aerobic glycolysis. Knockdown of UCPs and mTOR suppresses lactate production and increases ATP levels, suggesting that UCPs contribute to increased glycolysis. These results highlight the existence of a free radical-mediated mechanism that activates mitochondria uncoupling to reduce ROS production, which precedes the glycolytic adaptation described as the Warburg Effect.

Tumor necrosis factor alpha-mediated nitric oxide production enhances manganese superoxide dismutase nitration and mitochondrial dysfunction in primary neurons: an insight into the role of glial cells.

Tumor necrosis factor-alpha (TNF-alpha), a ubiquitous pro-inflammatory cytokine, is an important mediator in the immune-neuroendocrine system that affects the CNS. The present study demonstrates that treatment with TNF-alpha activates microglia to increase TNF-alpha production in primary cultures of glial cells isolated from wild-type (WT) mice and mice deficient in the inducible form of nitric oxide synthase (iNOSKO). However, mitochondrial dysfunction in WT neurons occurs at lower concentrations of TNF-alpha when neurons are directly treated with TNF-alpha or co-cultured with TNF-alpha-treated microglia than iNOSKO neurons similarly treated. Immunofluorescent staining of primary neurons co-cultured with TNF-alpha-treated microglia reveals that the antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), is co-localized with nitrotyrosine in WT but not in iNOSKO primary neuronal cells. Importantly, the percentage of surviving neurons is significantly reduced in WT neurons compared with iNOSKO neurons under identical treatment conditions. Together, the results suggest that TNF-alpha activates microglia to produce high levels of TNF-alpha and that production of nitric oxide (NO) in neurons is an important factor affecting MnSOD nitration and subsequent mitochondrial dysfunction.

RelB regulates manganese superoxide dismutase gene and resistance to ionizing radiation of prostate cancer cells.

The relationship between NF-kappaB and resistance to radiation treatment in many tumor cell types has been generally well recognized. However, which members of the NF-kappaB family contribute to radiation resistance is unclear. In the present study, we demonstrate that RelB plays an important radioprotective role in aggressive prostate cancer cells, in part by the induction of antioxidant and antiapoptotic manganese superoxide dismutase (MnSOD) gene. RelB is both constitutively present and is inducible by radiation in aggressive prostate cancer cells. Using ectopically expressed dominant negative inhibitor, p100 mutant, and the siRNA approach, we demonstrate that selective inhibition of RelB significantly decreases the levels of MnSOD resulting in a significant increase in the sensitivity of prostate cancer cells to radiation treatment. These results demonstrate that RelB plays an important role in redox regulation of the cell and protects aggressive prostate cancer cells against radiation-induced cell death. Thus, inhibition of RelB could be a novel mechanism to radiosensitize prostate cancer.

Advantage of protons compared to conventional X-ray or IMRT in the treatment of a pediatric patient with medulloblastoma.

PURPOSE: To compare treatment plans from standard photon therapy to intensity modulated X-rays (IMRT) and protons for craniospinal axis irradiation and posterior fossa boost in a patient with medulloblastoma. METHODS: Proton planning was accomplished using an in-house 3D planning system. IMRT plans were developed using the KonRad treatment planning system with 6-MV photons. RESULTS: Substantial normal-tissue dose sparing was realized with IMRT and proton treatment of the posterior fossa and spinal column. For example, the dose to 90% of the cochlea was reduced from 101.2% of the prescribed posterior fossa boost dose from conventional X-rays to 33.4% and 2.4% from IMRT and protons, respectively. Dose to 50% of the heart volume was reduced from 72.2% for conventional X-rays to 29.5% for IMRT and 0.5% for protons. Long-term toxicity with emphasis on hearing and endocrine and cardiac function should be substantially improved secondary to nontarget tissue sparing achieved with protons. CONCLUSION: The present study clearly demonstrates the advantage of conformal radiation methods for the treatment of posterior fossa and spinal column in children with medulloblastoma, when compared to conventional X-rays. Of the two conformal treatment methods evaluated, protons were found to be superior to IMRT.

Outcomes of an ambulatory cardiac pacing program: indications, risks, benefits, and outcomes.

UNLABELLED: The purpose of this review was to evaluate the economical benefits and outcomes of an ambulatory pacing system implantation program that began in 1991 for new and replacement pacing system implantations. Patient access, incidence of complications, hospital bed day utilization, economic impact, safety, and practicality of the Ambulatory Pacing System Implantation Program was retrospectively reviewed from August 1991 to March 1994. The Ambulatory Pacing System Implantation Program includes pre-operative outpatient assessment in the pacemaker clinic, admission to the Day Medical Unit followed by same day pacing system implantation, discharge home after a short period of observation, and, in selected cases, cardiac monitoring, followed by pacemaker clinic visit 24-hours postimplantation. RESULTS: Two hundred four cardiac pacing systems, 154 new and 50 replacements (pulse generator/leads), were implanted under the guidelines of the Ambulatory Pacing System Implantation Program between August 1991 and March 1994. Eighty-seven percent of the patients were implanted with passive fixation leads, the remaining with active fixation leads; all but four of which were leads implanted in the atria. Preprocedure wait time was decreased from 16 to 2 days for elective pacing system implantation. The incidence of lead dislodgment in the first, second, and third year of the Ambulatory Pacing System Implantation Program was 20%, 12%, 5%, respectively; compared to 11% for a similar number of cases in previous years done as inpatients. The incidence of new infection was 2.4% compared to < 1% in previous years. Six of the 204 (2%) cases required admission overnight. A total savings of 1,456 bed days ($1,275,450.00; Canadian) has been realized for an equivalent number of cases when compared with previous years. The operational cost was reduced by $3,976.00 per case. CONCLUSION: It has been shown that the Ambulatory Pacing System Implantation Program has improved client access, and is a safe and economical approach to implantation of cardiac pacing systems.

Expression of manganese superoxide dismutase promotes cellular differentiation.

Manganese superoxide dismutase (MnSOD) is a nuclear encoded mitochondrial matrix enzyme that scavenges toxic superoxide radicals. It has been shown that increased generation of reactive oxygen species is associated with the differentiation of microorganisms. To test the hypothesis that the ability of mitochondrial superoxide dismutase to neutralize a cellular hyperoxidant state is important for differentiation of mammalian cells, we examined the effect of transfection of MnSOD into mouse embryo fibroblasts on cellular differentiation. C3H10T1/2 cells served as a model for differentiation because these cells can be triggered to differentiate into myoblasts, adipocytes, and chondrocytes by treatment with 5-azacytidine. In this report, myoblast differentiation was defined by the presence of multinucleated cells, appearance of Z-bands, and expression of actin and desmin in the differentiated cells. Transfection of MnSOD gene was found to greatly enhance differentiation of C3H10T1/2 cells into myoblasts by 5-azacytidine. This result identifies MnSOD as an important factor for cell differentiation and supports a role for reactive oxygen species in the process of cellular differentiation.

Increased chronic bowel complications with split-course pelvic irradiation.

PURPOSE: To assess the possible impact of various treatment factors including split-course versus continuous course treatment on the incidence of chronic bowel complications in patients receiving adjuvant pelvic radiotherapy. METHODS AND MATERIALS: A retrospective review was performed of records of 153 patients treated with adjuvant external beam pelvic radiation therapy without brachytherapy for endometrial and colorectal carcinomas. Continuous course radiotherapy was administered in 91 patients (59%) and 62 patients (41%) received split course treatment with a planned 2 week mid-treatment break. Mean pelvic dose and daily fraction size were 51.4 and 1.71 Gray, respectively. Multiple patient and treatment variables were assessed for their possible relationship to chronic bowel complications. Univariate and multivariate statistical analyses were carried out. RESULTS: Twenty-seven patients (18%) developed chronic bowel complications at a median interval of 12 months after radiotherapy. Of all factors analyzed, only the use of split course technique was associated with a significantly higher rate of chronic bowel injury and decreased complication-free survival (p = 0.009). CONCLUSION: This study supports earlier suggestions that the use of split course rather than continuous course pelvic radiotherapy can increase late intestinal complication rates. Possible pathophysiologic mechanisms are discussed.

Distribution of the Bowman Birk protease inhibitor in mice following oral administration.

In this study, the distribution of the soybean-derived Bowman Birk inhibitor (BBI) in mice was examined. Mice received [125I]BBI by oral gavage and three hours later, the mice were sacrificed, the organs of interest were carefully removed and the distribution of the inhibitor was determined. The bulk of labeled BBI was present in the luminal contents of the small and large bowel, urine and feces. Significant amounts of label were also observed in the serum, esophagus, stomach and intestine, kidney, liver and lung. Analysis of tissue homogenates by gel filtration chromatography revealed that the radioactivity eluted from the column at the same position as the BBI standard indicating that the iodinated BBI was still intact. The chromatographically purified BBI was able to inhibit chymotrypsin indicating that functional protease inhibitory activity was present. These results indicate that the BBI becomes widely distributed in mice 3 h after oral administration and that intact protease inhibitor is present in internal organs.

Suppression of radiation-induced neoplastic transformation by overexpression of mitochondrial superoxide dismutase.

Manganese superoxide dismutase (MnSOD) scavenges toxic superoxide radicals produced in the mitochondria. Transfection of the human MnSOD gene into mouse C3H 10T1/2 cells resulted in production of active MnSOD, which was properly transported into mitochondria. Overexpression of MnSOD protected cells from radiation-, but not chemically-induced neoplastic transformation. This finding demonstrates that oxidative stress that occurs in the mitochondria plays an important role in the development of neoplastic transformation.

Influence of proliferation on DNA repair rates in liver.

To test the hypothesis that the proliferative status of a mammalian cell determines the rate of removal of oxidative DNA damage, pre- and posthepatectomized livers in adult male Fisher 344 rats were irradiated in situ with 15.5 Gy of 137Cs-gamma-rays. At 10 and 45 min after irradiation, the livers were removed and dissociated into single cell suspensions, and the DNA damage in the isolated quiescent or proliferative liver cells was assayed by alkaline elution. Proliferative liver cells irradiated 20-24 h or 29-31 h after hepatectomy repaired their DNA damage faster than quiescent liver cells. A corresponding increase in the accessibility of the DNA to digestion by m. nuclease was observed for the post-hepatectomized liver cells. These data suggest that proliferative status is a major determinant of the rate of DNA repair in rat liver.

Effect of the Bowman-Birk protease inhibitor on the expression of oncogenes in the irradiated rat colon.

In this study, we tested the influence of i.p. Bowman-Birk protease inhibitor (BBI) administration on oncogene expression in unirradiated and irradiated rat colonic mucosa. Total cellular RNA was collected from the colonic mucosa, and the levels of c-myc, c-fos, c-Ha-ras, c-EGFR, and c-actin mRNA were examined by standard dot and Northern blot analyses. The data demonstrate that BBI is capable of preventing radiation-induced overexpression of c-myc and c-fos without interfering with the constitutive expression of these 2 genes. It was also determined that BBI did not interfere with either radiation-induced overexpression of c-Ha-ras and c-EGFR or the constitutive expression of c-Ha-ras, c-EGFR, or c-actin. The data demonstrate that the anticarcinogenic BBI selectively inhibits the overexpression of c-myc and c-fos while not affecting crypt cell proliferation. These results suggest that a protease is involved in the pathway for enhanced c-myc and c-fos expression and that protease inhibitors such as BBI can interrupt this pathway.

Suppression of 3-methylcholanthrene-induced cellular transformation by timed administration of the Bowman-Birk protease inhibitor.

The Bowman-Birk protease inhibitor (BBI) is a legume-derived inhibitor of chymotrypsin and trypsin that has been shown to suppress cellular transformation and tumorigenesis. In the present investigation the effects of various BBI administration schedules were evaluated for suppression of 3-methylcholanthrene (3-MCA)-induced transformation of C3H/10T1/2 cells. At a concentration of 30 micrograms/ml, BBI demonstrated no toxicity to C3H/10T1/2 cells treated with 3-MCA. However, transformation of C3H/10T1/2 cells was significantly reduced when BBI was added to the cultures for a period of 14 or 42 days, starting immediately after exposure to the carcinogen. When BBI was administered only during the time of carcinogen exposure or alternatively beginning on day 15 and then continuously throughout the remainder of the 6-week transformation assay, it was ineffective for suppressing 3-MCA-induced cellular transformation. These findings indicate that BBI exerts its chemopreventive effect during the early stage of chemical carcinogen-induced cellular transformation.

The effects of the Bowman-Birk protease inhibitor on c-myc expression and cell proliferation in the unirradiated and irradiated mouse colon.

The levels of c-myc RNA and crypt cell proliferation were monitored in the mouse colonic mucosa following X-irradiation with and without oral administration of the Bowman-Birk protease inhibitor (BBI). Mice were divided into 4 groups and treated as follows: (A) daily gavage with water; (B) daily gavage with BBI; (C) daily gavage with water and 12 Gy of abdominal irradiation 1 day after the first gavage; (D) daily gavage with BBI and 12 Gy of abdominal irradiation 1 day after the first gavage. Samples were collected at various times after X-irradiation and ascending colon samples were taken for crypt cell proliferation analysis. The mucosa was removed from the remaining sample and total cellular RNA was isolated. The levels of c-myc mRNA underwent a time-dependent change following X-irradiation. Expression of the c-myc gene was unchanged at 1 day and 3 weeks after irradiation, but was markedly elevated at 1 week after X-irradiation. BBI was found to suppress the radiation induced elevation of c-myc mRNA, while having no effect on the rate of crypt cell proliferation or body weight of the mice.

Suppression of dimethylhydrazine-induced carcinogenesis in mice by dietary addition of the Bowman-Birk protease inhibitor.

In the present study the effect of feeding the soybean-derived Bowman-Birk protease inhibitor (BBI) on dimethylhydrazine (DHM)-induced gastrointestinal tract and liver carcinogenesis in mice was examined. In this investigation we found the addition of 0.5 or 0.1% semipurified BBI or 0.1% purified BBI to the diet of DMH-treated mice resulted in a statistically significant suppression of angiosarcomas and nodular hyperplasia of the liver and adenomatous tumors of the gastrointestinal tract. Autoclaved BBI or BBI which had its trypsin inhibitory domain specifically inactivated was found to be ineffective in suppressing the induction of these liver and gastrointestinal tract lesions. The results of this study also indicate that BBI, included as 0.5% of the diet or less, has the ability to suppress carcinogenesis with no observed adverse effects on the health of the mice.

Potential intracellular target proteins of the anticarcinogenic Bowman Birk protease inhibitor identified by affinity chromatography.

The soybean-derived Bowman Birk inhibitor (BBI) has been shown to inhibit carcinogenesis in both in vitro and in vivo model systems. In the present study, we have utilized a BBI affinity column to determine whether cellular enzymes, present in C3H/10T1/2 cells, specifically interact with this inhibitor. Using this technique, we have identified three proteins with masses of about 70, 60, and 50 kilodaltons. Cell fractionation experiments demonstrate that the 60- and 50-kilodalton proteins are present in the 10,000 x g pellet (lysosomal/golgi fraction) of C3H/10T1/2 cell homogenates. We have also identified two proteins with masses of 60 and 50 kilodaltons which bind to the BBI affinity column in fibroblasts from patients having Bloom syndrome. BBI as well as several other protease inhibitors has been shown previously to reduce the frequency of spontaneous chromosomal aberrations in these cells. Our results indicate that the 50- and 60-kilodalton proteins we have identified by affinity chromatography are present in both mouse and human cells and further suggest that these proteins are potential intracellular targets of the BBI in these cells.

Inhibition of radiation-induced transformation of C3H/10T1/2 cells by chymotrypsin inhibitor 1 from potatoes.

In this report, we have investigated whether a protease inhibitor obtained from potatoes (chymotrypsin inhibitor 1; CI-1) will inhibit carcinogen-induced transformation of C3H/10T1/2 cells. CI-1 was as effective as the soybean-derived Bowman Birk inhibitor at suppressing radiation-induced transformation of C3H/10T1/2 cells, at a concentration of 10 micrograms/ml. The inhibitor was not toxic to the cells at concentrations of 0.1-10 micrograms/ml, the concentrations of CI-1 employed in the transformation experiments. To investigate the interaction of this inhibitor with the target cells, binding studies were carried out. 125I-labelled CI-1 could not be displaced from C3H/10T1/2 cells by co-incubation of the cells with a 5000-fold excess of unlabelled inhibitor. These results suggest that this inhibitor does not reversibly bind to specific receptor proteins on the surface of these cells.

Crypt fission and crypt number in the small and large bowel of postnatal rats.

The aim of this investigation was to study crypt fission, a process which may be instrumental in regulating crypt number in the intestine. Young Holtzman rats were killed at various times after parturition and samples of the small intestine and colon were removed and processed. A microdissection technique was used to separate crypts from other structures. Crypts were scored as normal or fissioning. The percentage of crypts in fission (PCF) reached peak values of 25% and 52% in the small bowel and colon, respectively, at 21 days post-parturition. From this time onward, the PCF dropped until the adult value of approximately 7% was reached in each site. During this same period, the number of crypts increased from 1.9 X 10(6) to 3.3 X 10(6) in the small bowel and 2.2 X 10(5) to 6.5 X 10(5) in the colon. Thus an inverse relationship between the percentage of crypts in fission and crypt number was found. Distribution of fissure heights in fissioning crypts did not change as the animal aged. The majority of the fissures were found in the lower 1/4 of the fissioning crypts. This suggests that as soon as the fissure extends beyond the stem cell zone, division into two crypts soon occurs.

Response of cultured IEC-17 normal rat intestinal epithelial cells to X radiation.

The growth parameters and radiosensitivity of normal rat intestinal epithelial cells, IEC-17, were studied. The cells were cultured by standard methods and exposed to an array of doses (1-12 Gy) of 250 kVp X rays. The survival curves generated exhibited no initial shoulder and were bimodal. The Do of the first component was about 0.2 Gy and the second component. 5.0 Gy. The ability of this cell line to repair sublethal lesions was examined by fractionation studies; repair was completed within 60 min after the first dose. When Chinese hamster ovary (CHO) cells were grown under the same conditions used for the IEC-17 cells and then irradiated with single doses, a typical survival curve with a Do of 1.4 Gy was obtained. The survival curves obtained for the IEC-17 cell line are consistent with the response of a morphologically distinct single population containing two functionally separate types of cells.