Manganese superoxide dismutase deficiency enhances cell turnover via tumor promoter-induced alterations in AP-1 and p53-mediated pathways in a skin cancer model

Abstract

Previous studies in our laboratories demonstrated that overexpression of manganese superoxide dismutase (MnSOD) suppressed both the incidence and multiplicity of papillomas in a DMBA/TPA multi-stage skin carcinogenesis model. The activity of activator protein-1 (AP-1), which is associated with tumor promotion, was reduced in MnSOD transgenic mice overexpressing MnSOD in the skin, suggesting that MnSOD may reduce tumor incidence by suppressing AP-1 activation. In the present study, we report that reduction of MnSOD by heterozygous knockout of the MnSOD gene (Sod2 7/+, MnSOD KO) increased the levels of oxidative damage proteins and the activity of AP-1 following TPA treatment. RNA levels of ornithine decarboxylase (ODC) were also increased, suggesting an increase in cell proliferation in the KO mice. Histological examination con®rmed that the number of proliferating cells in DMBA/TPA-treated mouse skin were higher in the KO mice. Interestingly, histological examination also demonstrated greater numbers of apoptotic cells in the KO mice after DMBA/TPA treatment. Evidence of apoptosis, including DNA fragmentation, cytochrome c release from mitochondria, and caspase 3 activation were also observed by biochemical assays of the skin tissues. Apoptosis was associated with an increase in nuclear levels of p53 as determined by Western analysis. Quantitative immunogold ultrastructural analysis con®rmed that p53 immunoreactive protein levels were increased to a greater level in the nuclei of epidermal cells from MnSOD KO mice compared to epidermal nuclei from wild type mice similarly treated. Moreover, p53 levels further increased in the mitochondria of DMBA/TPA treated mice, and this increase was much greater in the MnSOD KO than in the wild type mice, suggesting a link between MnSOD de®ciency and mitochondrial-mediated apoptosis. Pathological examination reveals no dierence in the incidence and frequency of papillomas comparing the KO mice and their wild type littermates. Taken together, these results suggest that: (1) MnSOD de®ciency enhanced TPAinduced oxidative stress and AP-1 and p53 levels, consistent with the increase in both proliferation and apoptosis events in the MnSOD KO mice, and (2) increased apoptosis may negate increased proliferation in the MnSOD de®cient mice during an early stage of tumor development.

A Mechanism-Based Antioxidant Approach for the Reduction of Skin Carcinogenesis

“…Furthermore, studies with MnSOD-deficient mice confirmed and extended our finding on the role of MnSOD in suppressing AP-1 activity and provided important insights into the delicate relationship between the role of MnSOD in proliferation and apoptosis in an early stage of tumor development (13). The results showed that although cell turnover is drastically altered in MnSOD KO mice following DMBA/TPA treatment, resultant tumor formation is not changed, suggesting a tight regulation of apoptosis and proliferation in the mouse skin.…”

“…The cytosolic fraction was prepared as previously described (12,13). Skin epidermal cells were stripped and extracted by homogenization in 1 mL of homogenization buffer [20 mmol/L HEPES (pH 7.0), 5 mmol/L EGTA, 10 mmol/L 2-mercaptoethanol, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 Ag/mL each of protein inhibitors].…”

“…It is well recognized that DMBA or TPA treatment can cause oxidative stress (18,19). We have previously shown that TPA application induced rapid oxidative stress at the subcellular level as indicated by increased formation of carbonyl groups (aldehydes and ketones), a standard biomarker of oxidative damage, which are formed in the amino side chains of proteins (13). To verify the antioxidant potential of MnTE-2-PyP 5+ , whereas applied to mouse skin, we applied MnTE-2-PyP 5+ (5 ng/mouse) 30 minutes before TPA treatment, and protein carbonyl adducts were determined by Western blot.…”

“…These results suggested that MnSOD modulates tumor formation by inhibition of activator protein-1 (AP-1) signaling, resulting in reduction of tumors in MnSOD overexpressing mice. Surprisingly, in a MnSOD heterozygous knockout mouse model (MnSOD KO), there was no difference in the incidence and frequency of papillomas when comparing the MnSOD KO mice with their wild-type (WT) littermates (13). However, histologic examination showed that the number of proliferating cells in DMBA/TPAtreated mouse skin were higher in the KO mice.…”

Manganese superoxide dismutase and inducible nitric oxide synthase modify early oxidative events in acute Adriamycin-induced mitochondrial toxicity

“…Whole tissue homogenates were electrophoresed using standard SDS-PAGE techniques and used to detect MnSOD and CuZnSOD immunoreactive proteins by Western blot assay as previously described (14). Primary rabbit anti-MnSOD antibody (1:5,000) was purchased from Upstate Biotechnology (Lake Placid, NY) and primary sheep anti-CuZnSOD antibody (1:1,000) was purchased from Calbiochem (San Diego, CA).…”