Deregulated manganese superoxide dismutase expression and resistance to oxidative injury in p53-deficient cells.

Loss of function of the tumor suppressor protein p53 represents a very frequent event in human carcinogenesis, but the molecular mechanisms linking impaired p53 activity to increased cell malignancy are still incompletely understood. p53 is normally involved in both cell cycle control and the induction of cell death and is involved in the latter mainly through the transcriptional regulation of pro- and antiapoptotic proteins. Reactive oxygen species are known to be powerful inducers of p53 activity; moreover, they play a role in the execution of p53-dependent apoptosis. Here we show that transformed mouse fibroblasts lacking p53 are significantly more resistant than wild-type (wt) controls to the cytotoxic effect of a number of pro-oxidant treatments. Interestingly, these cells also exhibit deregulated expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD), a protein known to protect cancer cells from the oxidative injury inflicted by antitumoral cytokines and anticancer drugs. MnSOD activity was also increased in liver tissue from p53-deficient mice in comparison with wt tissue. Transient transfection of wt p53 in HeLa cells led to a significant reduction in steady-state MnSOD mRNA levels and enzymatic activity, confirming that the expression of this antioxidant enzyme is negatively regulated by p53. Forced expression of MnSOD rendered HeLa cells resistant to p53-dependent cytotoxic treatments and, in cotransfection experiments, counteracted the growth-inhibitory effect of p53. Taken together, these data identify MnSOD as a potential target for tumor suppressor protein p53 and underscore the relevance of MnSOD modulation in the context of normal p53 functions because it is consistent with many reports of abnormally increased MnSOD expression in human cancers.

Regulation of manganese superoxide dismutase (MnSOD) in chronic experimental alcoholism: effects of vitamin E-supplemented and -deficient diets.

In order to investigate the pathogenic mechanism responsible for liver injury associated with chronic alcoholism, we studied the effects of different dietary vitamin E levels in chronically ethanol (EtOH)-fed rats on the activity and mRNA regulation of the manganese superoxide dismutase (MnSOD) enzyme. Evidence is accumulating that intermediates of oxygen reduction may in fact be associated with the development of alcoholic liver disease. Since low vitamin E liver content seems to potentiate EtOH-linked oxidative stress, we studied the effect of EtOH treatment in livers from rats fed a diet deficient or supplemented with vitamin E. Chronic EtOH feeding enhanced hepatic consumption of vitamin E in both groups of EtOH-treated animals, irrespectively of the vitamin E level of the basal diet and the effect was observed in both the microsomal and mitochondrial fractions. Both EtOH-fed groups exhibited increased MnSOD gene expression, while the enzyme activity was enhanced only in the vitamin E-deprived group of EtOH-treated animals. The significant increase in manganese liver content found only in this last group could explain the rise of enzyme activity. In fact, in the absence of a parallel increase of the prosthetic ion manganese, MnSOD mRNA induction was not accompanied by a higher enzymatic activity. These findings support the role of oxidative alteration in the EtOH-induced chronic hepatotoxicity in which MnSOD response might represent a primary defence mechanism against the damaging effect of oxygen radical species.

Increased growth capacity of cervical-carcinoma cells over-expressing manganous superoxide dismutase.

Increases in the expression of manganese-dependent superoxide dismutase (MnSOD) have been detected in several classes of human and experimental tumors and appear to correlate with poorer prognosis in human neuro-epithelial, ovarian and cervical tumors. To delineate the relevance of MnSOD expression to tumor-cell growth and survival, a human MnSOD cDNA was over-expressed in the HeLa cervical-carcinoma cell line. MnSOD over-expression had marginal effects on the growth of HeLa cells in standard medium, but markedly protected the cells from growth suppression and cell death in conditions of serum deprivation. Serum starvation did not affect expression of endogenous MnSOD in wild-type HeLa cells, but was associated with increases in cell death and in the generation of intracellular oxygen radicals. By contrast, in HT29 colon-carcinoma cells, which are relatively resistant to growth-factor withdrawal, serum deprivation was associated with increases in MnSOD expression and activity. Together these observations suggest that MnSOD provides a mechanism for counteracting the intracellular oxidative processes that impair cell growth and viability in the context of growth-factor withdrawal and, in this context, may promote tumor-cell survival in vivo in conditions normally unfavorable to cell growth.

Quantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer.

Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.

Oxidative stress and overexpression of manganese superoxide dismutase in patients with Alzheimer's disease.

Substantial evidence supports the hypothesis that oxygen free radicals are involved in various neurodegenerative disorders. To assess the presence of oxidative stress in Alzheimer's disease (AD) we examined the activity of the enzyme copper-zinc superoxide dismutase (CuZnSOD) in red blood cells, the levels of the mitochondrial inducible enzyme manganese superoxide dismutase (MnSOD) mRNA in lymphocytes, and the total radical-trapping antioxidant capacity (TRAP) in plasma of AD patients and in a group of age-matched non-demented controls. We found that CuZnSOD activity (P < 0.01 vs. controls) was significantly increased as well as the MnSOD mRNA levels while the total antioxidant status (P < 0.001 vs. controls) was decreased in AD patients. These findings support the role of oxidative alterations in the pathogenetic mechanism underlying AD neurodegeneration.

Iron modulation of LPS-induced manganese superoxide dismutase gene expression in rat tissues.

Mitochondrial superoxide dismutase (MnSOD) is usually diminished in cancer cells. We observed that in vivo treatment with LPS produces a strong increase of MnSOD mRNA levels and a weak induction of an inactive protein in rat hepatocarcinomas. In normal liver iron deficiency, obtained with desferrioxamine administration, produces a decrease in the MnSOD induction by LPS, indicating that such induction could depend on tissue iron content. However, no change in MnSOD mRNA has been observed in iron-overloaded tumor tissue. Thus, iron is possibly involved in the transcriptional regulation of the protein, in combination with some other unknown factor that appears to be deficient in tumor cells.

The level of MnSOD is directly correlated with grade of brain tumours of neuroepithelial origin.

The oxy-radical scavenger enzyme manganese superoxide dismutase (MnSOD) may act in the capacity of a tumour-suppressor gene. To address the issue of its role in tumour transformation and progression in vivo, we evaluated the content of this enzyme in 33 brain tumours of neuroepithelial origin with different degrees of differentiation (WHO grade II-IV) by means of Western blot and immunohistology. Our results show that immunoreactive MnSOD increases in a direct relationship with tumour grade and is therefore inversely correlated with differentiation. The increase in induced at a pretranscriptional level and is apparently specific to brain tumours of neuroepithelial origin. Approximately 30% of grade IV tumours display low levels of MnSOD content, and preoperative radiotherapy and brachytherapy result in low amounts of enzyme. Based upon these observations, we suggest that MnSOD cannot be considered a classical tumour-suppressor gene.

Diethyldithiocarbamate treatment up regulates manganese superoxide dismutase gene expression in rat liver.

In vivo experiments demonstrate that rat liver manganese-containing superoxide dismutase (MnSOD) is up-regulated at the transcriptional level following the inactivation of copper-zinc superoxide dismutase (CuZnSOD). CuZnSOD activity was inhibited by the administration of the copper chelating agent diethyldithiocarbamate (DDC). This CuZnSOD inactivation is likely associated with an intracellular oxidative stress. Indeed the antioxidant N-acetyl-cysteine (NAC) completely prevents the MnSOD mRNA up-regulation observed after DDC administration. Evidence is also provided that an approximately 50% diminution of the total iron content in the tissue, which follows the in vivo administration of the iron chelator desferrioxamine (DESF), reduces the amount of MnSOD induction achieved by DDC treatment. Both NAC and DESF significantly down-regulate MnSOD gene expression also in normal untreated rat liver. While the observed inhibitory effect of NAC in MnSOD mRNA up-regulation can be ascribed mainly to its antioxidant property, iron chelation could act with an antioxidant effect and/or affecting some iron-dependent factor(s) possibly involved in MnSOD gene regulation. It is proposed that this metal could have a role among factors that sense and/or trigger transcription of the MnSOD gene.