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Mechanism of the tumor suppressive effect of MnSOD overexpression
Biomedicine & Pharmacotherapy 59 (2005) 143–148 http://france.elsevier.com/direct/BIOPHA/
Dossier: Superoxide dismutases: recent advances and clinical applications
Mechanism of the tumor suppressive effect of MnSOD overexpression Larry W. Oberley * Free Radical and Radiation Biology, Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa City, Iowa 52242, USA Received 3 November 2004 Available online 19 March 2005
Abstract The mitochondrial antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been shown to be a new type of tumor suppressor protein. Overexpression of MnSOD protein inhibits growth in a wide variety of cancer types. This review examines the molecular mechanism of the tumor suppressive effect of MnSOD. Three species have been proposed to cause the tumor suppressive effect: superoxide radical, hydrogen peroxide and nitric oxide. At the present time, the evidence appears strongest that hydrogen peroxide is the effector molecule since both catalase and glutathione peroxidase has been shown to modulate the effect. Surprisingly, in different cancer cell lines, overexpression of GPx has been found to both decrease and increase the growth inhibitory effect of MnSOD overexpression. Knowledge of which molecule causes the tumor suppressive effect of MnSOD and the mechanism of action will likely lead to new therapies for the treatment of cancer. © 2005 Elsevier SAS. All rights reserved. Keywords: Tumor suppressor; Manganese-containing superoxide dismutase; Glutathione peroxidase; Catalase
1. Introduction The superoxide dismutase (SOD) family of proteins is necessary to protect oxygen-utilizing cells from the toxicity of the reactive oxygen species (ROS) produced during normal metabolism. Besides being protective proteins, these enzymes are also key components of signaling pathways that regulate •cell physiology. The SODs catalyze the reaction: 2O2 + + 2H →H 2 O 2 + O 2 . Hydrogen peroxide is then removed by catalases (CATs) and peroxidases, of which glutathione per-
Abbreviations: BCNU, 1.3 bis (2-chloroethyl)-1-nitrosourea; BSO, buthionine sulfoximine; CAT, catalase protein; CAT, catalase gene; CuZnSOD, copper- and zinc-containing superoxide dismutase protein; CuZnSOD, copper- and zinc-containing superoxide dismutase gene; ECSOD, extracellular superoxide dismutase protein; eNOS, endothelial nitric oxide synthase protein; GPx, glutathione peroxidase protein; GPx, glutathione peroxidase gene; GR, glutathione reductase protein; GSH, reduced glutathione; GSSG, glutathione disulfide; MnSOD, manganese-containing superoxide dismutase protein, MnSOD, manganese-containing superoxide dismutase gene; MOI, multiplicity of infection; mtCAT, mitochondrial form of catalase protein; NOS, nitric oxide synthase protein; NOS, nitric oxide synthase gene; ROS, reactive oxygen species; SOD, superoxide dismutase protein; TNF, tumor necrosis factor. * Corresponding author. Tel.: +1 319 335 8015; fax: +1 319 335 8039. E-mail address: [email protected] (L.W. Oberley). 0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2005.03.006
oxidase (GPx) has been the most widely studied. There are three known forms of SOD in mammalian cells: a copperand zinc-containing superoxide dismutase (CuZnSOD) found mainly in the cytoplasm and nucleus, a manganese-containing superoxide dismutase (MnSOD) found in the mitochondria, and an extracellular superoxide dismutase (ECSOD) found primarily in the extracellular compartments. The purpose of this review article is to discuss the role of MnSOD as a tumor suppressor protein and to suggest possible mechanisms for its tumor suppressive ability. It has been over 30 years now since the first report was published demonstrating that the activity of MnSOD was diminished in transformed cells when compared to an appropriate normal cell control [43]. Since that time, numerous papers have been published showing altered levels of antioxidant enzymes in cancer cells; this subject matter has been reviewed many times [27–32]. Cancer cells are nearly always low in MnSOD and catalase (CAT) activity, and usually low in CuZnSOD activity [27–32]. Glutathione peroxidase (GPx) activity is variable. Recently, it has been shown that in some cancer cells, reduced expression of MnSOD is due to mutations in the promoter of the gene [42], while in other types of cancer, reduced levels of MnSOD are due to abnormal methylation [11], loss of heterozygosity [26,18], or mutation in the
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coding sequence [10]. Thus, MnSOD loss is similar in mechanism to that reported for other tumor suppressor genes. Even though there is a large body of literature linking free radicals and antioxidant enzymes to cancer, most of the evidence is correlative and does not demonstrate a causal relationship. There are several lines of evidence that do imply a causal relationship. Powerful evidence for a causal relationship is that in various model systems, ROS cause cancer; moreover, antioxidants in general, and SOD and SODmimetics in particular, inhibit malignant transformation [6,29,31]. Molecular biological techniques have been also used to demonstrate an important role for SOD in transformation; overexpression of MnSOD by cDNA transfection led to inhibition of radiation-induced transformation in a mouse fibroblast cell line [37]. Recently, it has been shown that a life-long reduction in MnSOD activity (in transgenic heterozygotic mice with a 50% reduction in MnSOD activity) results in a much higher incidence of cancer [39].
7 cells [14], virally-transformed WI-38 human lung fibroblasts [44], A172R rat glioma [48], U118 human glioma [47], human oral squamous carcinoma SCC-25 [21], mouse [35,36] and human fibrosarcoma [25], human prostatic carcinoma DU145 [15], and human pancreatic cancer cells [40]. Therefore, in all these tumor types, overexpression of MnSOD led to suppression of at least part of the tumor cell phenotype. This work has been done at five different institutions (University of Iowa, Washington University, University of Kentucky, University of Wisconsin and Albany Medical College). Thus, the evidence appears substantial that MnSOD elevation by cDNA transfection can suppress the malignant phenotype in a great variety of tumors. On the basis of this work showing growth suppression and the fact that loss of heterozygosity (LOH) for MnSOD has been found in human melanoma [26] and glioma [18], we and others have proposed that MnSOD is a new type of tumor suppressor gene [5].
2. Effect of increasing SOD on the cancer phenotype
4. MnSOD enzymatic activity causes the tumor suppressive effect of MnSOD protein
If SODs are important in cancer, then normalization of the levels of these enzymes should result in reversal of at least part of the cancer cell phenotype. This hypothesis was first suggested by Oberley and Buettner [28] and has been tested with regards to SOD in three different ways: (1) elevation of SOD by exposure to a superoxide generator and subsequent isolation of resistant cells [9]; (2) addition of liposomal CuZnSOD protein [3] and (3) elevation of SOD, particularly MnSOD, by sense cDNA transfection. Each of these techniques has supported the Oberley–Buettner hypothesis. For brevity, only cDNA transfection will be discussed.
3. Increasing MnSOD by cDNA transfection The first paper using cDNA transfection of MnSOD was published in 1993 [7]. In collaboration with Drs. Sue Church and James Grant at Washington University, we demonstrated that the transfection of MnSOD cDNA into cultured human melanoma cells resulted in the loss of the malignant phenotype. The malignant phenotype was tested both in vitro by assays such as mitotic rate and growth in soft agar and, more importantly, in vivo by growth in nude mice. All of these tests showed a loss of the malignant phenotype in clones that overexpressed MnSOD by at least five-fold. The most important observations were that in the nude mouse assay, 18 out of 18 sites injected with the parental melanoma cell line developed tumors, while 0 out of 16 sites injected with high MnSOD overexpressing cells developed tumors. We and others have published papers on many other cancer cell types and one virally-transformed cell line showing that overexpression of MnSOD in each of these cell lines led to suppression of cell growth both in vitro and in vivo. Growth suppression was observed in human breast carcinoma MCF-
It has been reported that the MnSOD protein has two variants at amino acid 58; either isoleucine (Ile) or threonine (Thr) can be at this position in the protein [4]. It is still unclear whether this variation is a polymorphism or is a cancer mutation. Isolated Ile58 protein was found to possess twice the enzymatic activity of the Thr58 form and to be more stable against heat [4]. We sequenced the cDNA we had been transfecting and found it contained lesser activity Thr58 form. We used site directed mutagenesis to make the Ile58 form. We then transfected both forms into wild type MCF-7 cells and isolated overexpressing clones [45]. Four clones overexpressing Thr58 MnSOD and eight clones overexpressing Ile58 MnSOD were isolated and characterized. The Ile58 clones had three times the specific activity of the Thr58 form. Both forms of the MnSOD had tumor suppressive activity that was in general proportional to the MnSOD activity. The Ile58 clones had a higher tumor suppressive effect apparently because they had higher MnSOD activity. These results suggest that tumor suppressive effect of MnSOD is due to its enzymatic activity. In unpublished work, we have confirmed this observation with another MnSOD mutation that leads to partial inactivation. We believe our results have far reaching implications. A paper has recently appeared demonstrating that another polymorphism for MnSOD caused increased risk for breast cancer [2]. A valine or alanine can be at the –9 position in the MnSOD mitochondrial targeting presequence. Premenopausal women who were homozygous for the alanine allele had a 4-fold increase in breast cancer risk compared to those with 1 or 2 valine alleles. This suggests that MnSOD polymorphisms may be important in cancer susceptibility. It is very logical that individuals who have an MnSOD protein that is less active should be more susceptible to oxidative
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stress. Hence, we would predict that individuals that express the Thr58 form of MnSOD may be much more susceptible to cancer than those who express the Ile58 form. We are currently investigating this hypothesis.
5. Biological mechanism of MnSOD as a tumor suppressor We are also investigating the mechanism of the tumor suppression by MnSOD overexpression. Our studies to date have shown no evidence for necrosis, apoptosis and inflammatory events. In other words, we have found so far no evidence of cell death as a mechanism. We hypothesize that the effects of MnSOD overexpression on cancer cells are due to a noncytotoxic tumor suppressive effect. We have demonstrated changes in cell cycle parameters following MnSOD overexpression using flow cytometry [16]. Our working hypothesis at present is that MnSOD overexpression leads to changes in the superoxide/hydrogen peroxide balance and this causes changes in the redox state that affects signal transduction pathways modulating cell proliferation. This is a reasonable hypothesis because in the last several years, there has been an explosion of literature demonstrating that kinases, phosphatases and transcription factors are all redox-modulated by ROS [33,23,1,12,38]. We have shown that MnSOD overexpression leads to an inhibition of the AP-1 transcription factor and an activation of the NF-jB transcription factor in human breast cancer cells [13]. Our hypothesis is supported by the work in Gisela Storz’s laboratory at the NIH. They have shown that the OxyR transcription factor in Escherichia coli is activated through the formation of a disulfide bond and is deactivated by enzymatic reduction with glutaredoxin [46]. The OxyR transcription factor is sensitive to oxidation and activates the expression of genes in response to hydrogen peroxide. The redox potential of OxyR was determined to be –185 mV; since the redox potential of E. coli cytosol is –280 mV, OxyR is reduced in the absence of stress. These results indicate an example of redox signaling through disulfide bond formation and reduction. A similar example in E. coli is the Sox R transcription factor, which activates genes in response to superoxide radicals; Sox R is an iron–sulfur protein whose activation is redox-modulated through superoxide radical. Thus, in E. coli the redox potential of the cell is modulated by superoxide and hydrogen peroxide; the redox potential in turn governs the activity of these two transcription factors that regulate the expression of a large number of genes. We believe similar regulation occurs in mammalian cells, although the regulation is far more complex. In collaboration with Dr. Bharat Aggarwal, we have shown that MnSOD overexpression in mammalian cells suppresses tumor necrosis factor (TNF)-induced apoptosis and activation of NF-jB and AP-1 transcription factors [24]. Besides TNF, phorbol ester-, okadaic acid-, ceramide- and lipopolysaccharide-induced activation of NF-jB was blocked by MnSOD, indicating a common pathway of activation. In
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addition, MnSOD blocked the TNF-mediated activation of AP-1, stress-activated c-Jun protein kinase, and mitogenactivated protein kinase kinase. These results demonstrate that MnSOD overexpression can have a dramatic effect on signal transduction pathways.
6. Molecular species responsible for tumor suppressive effect of MnSOD So far, three species have been suggested as effectors for the MnSOD tumor suppressive effect: superoxide radical, hydrogen peroxide and nitric oxide. Superoxide radical and hydrogen peroxide are logical since they are the substrate and product of the superoxide dismutase enzymatic reaction, respectively. Nitric oxide radical is also logical since it reacts with superoxide with a rate constant comparable to that of MnSOD. Our work has focused on testing hydrogen peroxide as an effector, since there are enzymes that specifically remove this species. Thus, we have already transfected cytosolic glutathione peroxidase (GPx1) into MnSOD overexpressing cells and demonstrated a modulation of the tumor suppressive effect in all cell lines tested. Surprisingly, in one cancer cell line, GPx1 overexpression caused an inhibition of the tumor suppressive effects of MnSOD [17], while in another cell line GPx1 overexpression actually increased the tumor suppressive effect of MnSOD [19]. A major goal of our future work will be to understand why different results are obtained in different cell lines. Since GPx1 can also remove other hydroperoxides, it is also unclear if its effect is due to hydrogen peroxide or other hydroperoxides. For this reason, we also plan in the future to transfect both peroxisomal and mitochondrial catalase (made by adding the MnSOD presequence) into MnSOD overexpressing cells to determine the effect. Catalase does not effectively act on lipid hydroperoxides, but does remove hydrogen peroxide effectively. Recently, Rodriguez et al. [34] have shown that either peroxisomal or mitochondrial plus peroxisomal catalase can reverse the MnSOD-dependent inhibition of cell growth and plating efficiency seen in a human fibrosarcoma cell line. These results suggest that hydrogen peroxide is involved in the tumor suppressive effects seen with MnSOD and this work with catalase overexpression needs to be reproduced in other transfected cell lines. Very recently, work has been published that supports a strong role for hydrogen peroxide, in the growth suppressive effect of MnSOD [8]. Davis et al. [8] have shown potent growth inhibitory effects of an active site mutant of MnSOD that has increased enzymatic efficiency. His-30 of MnSOD is part of a hydrogen-bonding network in the active site, which may be important for the delivery of protons to regenerate the active Mn(III) enzyme. Davis et al. [8] made a H30 N (substitution of asparagine at His-30) mutant that has greatly increased catalytic efficiency. The authors tried to make stable H30 N overexpressing cell lines by plasmid transfection of HEK 293 cells, but were unable to do so because the selected
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cells would not grow. Over the time period necessary for stable selection, overexpression of the H30 N protein caused significant slowing of cell growth in multiple independent clones and separate transfections, irrespective of the nature of the vector or selection regimen. It was found that the levels of ROS measured by DCF-DA fluorescence were greatly increased in these cells. Since catalase reduced the ROS signal, it was hypothesized that hydrogen peroxide caused the antiproliferative effect of the mutant enzyme. To examine this hypothesis, clonal cell lines that stably overexpressed mitochondrial catalase (mtCAT) were first established; these lines were then doubly transfected with plasmids containing H30 N. As predicted, the inclusion of mtCAT eliminated the quiescent phenotype associated with H30 N transfection and suggested that hydrogen peroxide was responsible for the growth inhibition. The authors also showed the growth inhibitory effect of MnSOD overexpression using retroviral-mediated gene targeting of A549 human lung adenocarcinoma cells. This technique allowed transduction in a much smaller period of time. With this technique, H30 N overexpressing cells were isolated and grew much more slowly than control, vector transduced, or wild type (WT) MnSOD transduced cells. Immunoblot analysis showed similar amounts of immunoreactive protein in WT or H30 N-overexpressing cells, thus excluding the possibility that changes in MnSOD protein levels were responsible for the slower growth of the mutant cells. To test the antitumor effect of H30 N in vivo, A549 cells were transduced with a retroviral vector. Forty-eight hours following transduction, cells were injected subcutaneously into NOD/SCID mice and tumor volume was assessed with time. A great inhibition of tumor growth was observed in H30 N mice compared to those transduced with vector alone or WT MnSOD. The authors conclude that H30 N has a greatly increased growth inhibitory capacity due to its greater production of hydrogen peroxide. This study strongly supports the hypothesis that the growth inhibition caused by MnSOD overexpression is due to the hydrogen peroxide produced.
7. Effect of eNOS on the tumor suppressive effect of MnSOD Andres Melendez has suggested that nitric oxide is also involved in the suppression of cancer cell proliferation by MnSOD [25]. Nitric oxide is known to react with superoxide radical with a rate constant higher that of superoxide with MnSOD; thus, nitric oxide and MnSOD compete in cells for superoxide radical. Dr. Melendez found that MnSOD overexpression enhanced the cytostatic action of three nitric oxide donor compounds. Thus, nitric oxide enhanced the inhibition of cell proliferation caused by MnSOD overexpression. In the Melendez paper, the effect of MnSOD overexpression was lost at high concentrations of nitric oxide donors. Hence, nitric oxide concentrations may determine the results. More work is necessary to examine the possibility of nitric oxide involvement. If neither CAT nor GPx nor NOS affected the tumor
suppressive effect of MnSOD, we could conclude that the sole effector molecule was superoxide radical; the above data does not support this possibility in the few cell lines examined. We have also examined the effect of nitric oxide by transfecting the endothelial nitric oxide synthase gene (eNOS) into a human oral cancer SCC-25 cell line [22,20]. We found that overexpression of eNOS inhibited oral cancer growth in vitro, but in vivo the transfectants grew faster than wild type or vector controls [22]. So different results were obtained in vitro compared to in vivo. One possible explanation for these results is that NOS can under certain conditions produce not only nitric oxide, but also superoxide radical. Different species may have been produced by NOS in in vitro conditions compared to in vivo conditions. In addition, NO• can diffuse out of the transfected tumor cell, so that in vivo, effects on surrounding normal cells are also possible. In fact, increased normal cell proliferation after eNOS transfection appeared to be present in our in vivo model [22]. We also previously transfected eNOS into an MnSOD overexpressing clone [20]. Little effect was seen in vitro, but in vivo eNOS overexpression actually inhibited about 50% the tumor suppressive effect of MnSOD. This work was not published because it was incomplete and must be further studied in the future.
8. Increased anticancer cytotoxicity with BCNU We have found that MnSOD in combination with certain chemicals can have an anticancer effect that causes cell killing in contrast to the non-cytotoxic tumor suppressive effect described above for MnSOD alone. The enzymatic effect of MnSOD protein is to dismute superoxide radical into hydrogen peroxide. If we inhibit hydrogen peroxide removal, then we should kill cancer cells because of direct toxicity or hydrogen peroxide-mediated damage. We have tested this idea in tissue culture using stable plasmid transfected rat glioma cells and found very positive results [48]. The higher the MnSOD levels, the higher the killing we observed in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or buthionine sulfoximine (BSO). BCNU is a clinically used anticancer drug that causes alkylation and also inhibits glutathione reductase (GR). If GR is inhibited, cells cannot remove hydrogen peroxide [48]. BSO is a competitive inhibitor of a glutathione synthesis protein. If BSO is given to cells, then glutathione is depleted and again hydrogen peroxide cannot be removed by the glutathione peroxidase system. As an example of the effectiveness of this combination, with wild type and vector control cells, about 10% of the cells were killed by 50 µM BSO, while 100% of the high MnSOD overexpressing cells were killed by the same dose of BSO [48]. These data again suggests that hydrogen peroxide is a mediator of the tumor suppressive effects of MnSOD. The significance of this observation is that MnSOD overexpression can be used as the basis for a new anticancer combination therapy. We have shown that adenoviral MnSOD when given with BCNU is a power-
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ful antitumor therapy in a mouse xenograft model of human oral cancer [41].
9. Significance The significance of this work is the understanding of the mechanism of the tumor suppressive effect of MnSOD. If we can determine the molecular species responsible for the tumor suppressive effect, then we can maximize this pathway to produce the largest effect on cancer cell growth. We are already developing therapies based on overexpression of MnSOD and the proposed work will help us maximize that therapy. An example of where we could go from this research is that if we determined that hydrogen peroxide caused the tumor suppressive effect of MnSOD, we could develop inhibitors of all the hydrogen peroxide removing molecules in the mitochondria of cancer cells to be given in combination with MnSOD overexpression. We feel that overexpression of MnSOD has real potential in cancer therapy.
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Dossier: Superoxide dismutases: recent advances and clinical applications
Mechanism of the tumor suppressive effect of MnSOD overexpression Larry W. Oberley * Free Radical and Radiation Biology, Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa City, Iowa 52242, USA Received 3 November 2004 Available online 19 March 2005
Abstract The mitochondrial antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been shown to be a new type of tumor suppressor protein. Overexpression of MnSOD protein inhibits growth in a wide variety of cancer types. This review examines the molecular mechanism of the tumor suppressive effect of MnSOD. Three species have been proposed to cause the tumor suppressive effect: superoxide radical, hydrogen peroxide and nitric oxide. At the present time, the evidence appears strongest that hydrogen peroxide is the effector molecule since both catalase and glutathione peroxidase has been shown to modulate the effect. Surprisingly, in different cancer cell lines, overexpression of GPx has been found to both decrease and increase the growth inhibitory effect of MnSOD overexpression. Knowledge of which molecule causes the tumor suppressive effect of MnSOD and the mechanism of action will likely lead to new therapies for the treatment of cancer. © 2005 Elsevier SAS. All rights reserved. Keywords: Tumor suppressor; Manganese-containing superoxide dismutase; Glutathione peroxidase; Catalase
1. Introduction The superoxide dismutase (SOD) family of proteins is necessary to protect oxygen-utilizing cells from the toxicity of the reactive oxygen species (ROS) produced during normal metabolism. Besides being protective proteins, these enzymes are also key components of signaling pathways that regulate •cell physiology. The SODs catalyze the reaction: 2O2 + + 2H →H 2 O 2 + O 2 . Hydrogen peroxide is then removed by catalases (CATs) and peroxidases, of which glutathione per-
Abbreviations: BCNU, 1.3 bis (2-chloroethyl)-1-nitrosourea; BSO, buthionine sulfoximine; CAT, catalase protein; CAT, catalase gene; CuZnSOD, copper- and zinc-containing superoxide dismutase protein; CuZnSOD, copper- and zinc-containing superoxide dismutase gene; ECSOD, extracellular superoxide dismutase protein; eNOS, endothelial nitric oxide synthase protein; GPx, glutathione peroxidase protein; GPx, glutathione peroxidase gene; GR, glutathione reductase protein; GSH, reduced glutathione; GSSG, glutathione disulfide; MnSOD, manganese-containing superoxide dismutase protein, MnSOD, manganese-containing superoxide dismutase gene; MOI, multiplicity of infection; mtCAT, mitochondrial form of catalase protein; NOS, nitric oxide synthase protein; NOS, nitric oxide synthase gene; ROS, reactive oxygen species; SOD, superoxide dismutase protein; TNF, tumor necrosis factor. * Corresponding author. Tel.: +1 319 335 8015; fax: +1 319 335 8039. E-mail address: [email protected] (L.W. Oberley). 0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2005.03.006
oxidase (GPx) has been the most widely studied. There are three known forms of SOD in mammalian cells: a copperand zinc-containing superoxide dismutase (CuZnSOD) found mainly in the cytoplasm and nucleus, a manganese-containing superoxide dismutase (MnSOD) found in the mitochondria, and an extracellular superoxide dismutase (ECSOD) found primarily in the extracellular compartments. The purpose of this review article is to discuss the role of MnSOD as a tumor suppressor protein and to suggest possible mechanisms for its tumor suppressive ability. It has been over 30 years now since the first report was published demonstrating that the activity of MnSOD was diminished in transformed cells when compared to an appropriate normal cell control [43]. Since that time, numerous papers have been published showing altered levels of antioxidant enzymes in cancer cells; this subject matter has been reviewed many times [27–32]. Cancer cells are nearly always low in MnSOD and catalase (CAT) activity, and usually low in CuZnSOD activity [27–32]. Glutathione peroxidase (GPx) activity is variable. Recently, it has been shown that in some cancer cells, reduced expression of MnSOD is due to mutations in the promoter of the gene [42], while in other types of cancer, reduced levels of MnSOD are due to abnormal methylation [11], loss of heterozygosity [26,18], or mutation in the
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coding sequence [10]. Thus, MnSOD loss is similar in mechanism to that reported for other tumor suppressor genes. Even though there is a large body of literature linking free radicals and antioxidant enzymes to cancer, most of the evidence is correlative and does not demonstrate a causal relationship. There are several lines of evidence that do imply a causal relationship. Powerful evidence for a causal relationship is that in various model systems, ROS cause cancer; moreover, antioxidants in general, and SOD and SODmimetics in particular, inhibit malignant transformation [6,29,31]. Molecular biological techniques have been also used to demonstrate an important role for SOD in transformation; overexpression of MnSOD by cDNA transfection led to inhibition of radiation-induced transformation in a mouse fibroblast cell line [37]. Recently, it has been shown that a life-long reduction in MnSOD activity (in transgenic heterozygotic mice with a 50% reduction in MnSOD activity) results in a much higher incidence of cancer [39].
7 cells [14], virally-transformed WI-38 human lung fibroblasts [44], A172R rat glioma [48], U118 human glioma [47], human oral squamous carcinoma SCC-25 [21], mouse [35,36] and human fibrosarcoma [25], human prostatic carcinoma DU145 [15], and human pancreatic cancer cells [40]. Therefore, in all these tumor types, overexpression of MnSOD led to suppression of at least part of the tumor cell phenotype. This work has been done at five different institutions (University of Iowa, Washington University, University of Kentucky, University of Wisconsin and Albany Medical College). Thus, the evidence appears substantial that MnSOD elevation by cDNA transfection can suppress the malignant phenotype in a great variety of tumors. On the basis of this work showing growth suppression and the fact that loss of heterozygosity (LOH) for MnSOD has been found in human melanoma [26] and glioma [18], we and others have proposed that MnSOD is a new type of tumor suppressor gene [5].
2. Effect of increasing SOD on the cancer phenotype
4. MnSOD enzymatic activity causes the tumor suppressive effect of MnSOD protein
If SODs are important in cancer, then normalization of the levels of these enzymes should result in reversal of at least part of the cancer cell phenotype. This hypothesis was first suggested by Oberley and Buettner [28] and has been tested with regards to SOD in three different ways: (1) elevation of SOD by exposure to a superoxide generator and subsequent isolation of resistant cells [9]; (2) addition of liposomal CuZnSOD protein [3] and (3) elevation of SOD, particularly MnSOD, by sense cDNA transfection. Each of these techniques has supported the Oberley–Buettner hypothesis. For brevity, only cDNA transfection will be discussed.
3. Increasing MnSOD by cDNA transfection The first paper using cDNA transfection of MnSOD was published in 1993 [7]. In collaboration with Drs. Sue Church and James Grant at Washington University, we demonstrated that the transfection of MnSOD cDNA into cultured human melanoma cells resulted in the loss of the malignant phenotype. The malignant phenotype was tested both in vitro by assays such as mitotic rate and growth in soft agar and, more importantly, in vivo by growth in nude mice. All of these tests showed a loss of the malignant phenotype in clones that overexpressed MnSOD by at least five-fold. The most important observations were that in the nude mouse assay, 18 out of 18 sites injected with the parental melanoma cell line developed tumors, while 0 out of 16 sites injected with high MnSOD overexpressing cells developed tumors. We and others have published papers on many other cancer cell types and one virally-transformed cell line showing that overexpression of MnSOD in each of these cell lines led to suppression of cell growth both in vitro and in vivo. Growth suppression was observed in human breast carcinoma MCF-
It has been reported that the MnSOD protein has two variants at amino acid 58; either isoleucine (Ile) or threonine (Thr) can be at this position in the protein [4]. It is still unclear whether this variation is a polymorphism or is a cancer mutation. Isolated Ile58 protein was found to possess twice the enzymatic activity of the Thr58 form and to be more stable against heat [4]. We sequenced the cDNA we had been transfecting and found it contained lesser activity Thr58 form. We used site directed mutagenesis to make the Ile58 form. We then transfected both forms into wild type MCF-7 cells and isolated overexpressing clones [45]. Four clones overexpressing Thr58 MnSOD and eight clones overexpressing Ile58 MnSOD were isolated and characterized. The Ile58 clones had three times the specific activity of the Thr58 form. Both forms of the MnSOD had tumor suppressive activity that was in general proportional to the MnSOD activity. The Ile58 clones had a higher tumor suppressive effect apparently because they had higher MnSOD activity. These results suggest that tumor suppressive effect of MnSOD is due to its enzymatic activity. In unpublished work, we have confirmed this observation with another MnSOD mutation that leads to partial inactivation. We believe our results have far reaching implications. A paper has recently appeared demonstrating that another polymorphism for MnSOD caused increased risk for breast cancer [2]. A valine or alanine can be at the –9 position in the MnSOD mitochondrial targeting presequence. Premenopausal women who were homozygous for the alanine allele had a 4-fold increase in breast cancer risk compared to those with 1 or 2 valine alleles. This suggests that MnSOD polymorphisms may be important in cancer susceptibility. It is very logical that individuals who have an MnSOD protein that is less active should be more susceptible to oxidative
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stress. Hence, we would predict that individuals that express the Thr58 form of MnSOD may be much more susceptible to cancer than those who express the Ile58 form. We are currently investigating this hypothesis.
5. Biological mechanism of MnSOD as a tumor suppressor We are also investigating the mechanism of the tumor suppression by MnSOD overexpression. Our studies to date have shown no evidence for necrosis, apoptosis and inflammatory events. In other words, we have found so far no evidence of cell death as a mechanism. We hypothesize that the effects of MnSOD overexpression on cancer cells are due to a noncytotoxic tumor suppressive effect. We have demonstrated changes in cell cycle parameters following MnSOD overexpression using flow cytometry [16]. Our working hypothesis at present is that MnSOD overexpression leads to changes in the superoxide/hydrogen peroxide balance and this causes changes in the redox state that affects signal transduction pathways modulating cell proliferation. This is a reasonable hypothesis because in the last several years, there has been an explosion of literature demonstrating that kinases, phosphatases and transcription factors are all redox-modulated by ROS [33,23,1,12,38]. We have shown that MnSOD overexpression leads to an inhibition of the AP-1 transcription factor and an activation of the NF-jB transcription factor in human breast cancer cells [13]. Our hypothesis is supported by the work in Gisela Storz’s laboratory at the NIH. They have shown that the OxyR transcription factor in Escherichia coli is activated through the formation of a disulfide bond and is deactivated by enzymatic reduction with glutaredoxin [46]. The OxyR transcription factor is sensitive to oxidation and activates the expression of genes in response to hydrogen peroxide. The redox potential of OxyR was determined to be –185 mV; since the redox potential of E. coli cytosol is –280 mV, OxyR is reduced in the absence of stress. These results indicate an example of redox signaling through disulfide bond formation and reduction. A similar example in E. coli is the Sox R transcription factor, which activates genes in response to superoxide radicals; Sox R is an iron–sulfur protein whose activation is redox-modulated through superoxide radical. Thus, in E. coli the redox potential of the cell is modulated by superoxide and hydrogen peroxide; the redox potential in turn governs the activity of these two transcription factors that regulate the expression of a large number of genes. We believe similar regulation occurs in mammalian cells, although the regulation is far more complex. In collaboration with Dr. Bharat Aggarwal, we have shown that MnSOD overexpression in mammalian cells suppresses tumor necrosis factor (TNF)-induced apoptosis and activation of NF-jB and AP-1 transcription factors [24]. Besides TNF, phorbol ester-, okadaic acid-, ceramide- and lipopolysaccharide-induced activation of NF-jB was blocked by MnSOD, indicating a common pathway of activation. In
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addition, MnSOD blocked the TNF-mediated activation of AP-1, stress-activated c-Jun protein kinase, and mitogenactivated protein kinase kinase. These results demonstrate that MnSOD overexpression can have a dramatic effect on signal transduction pathways.
6. Molecular species responsible for tumor suppressive effect of MnSOD So far, three species have been suggested as effectors for the MnSOD tumor suppressive effect: superoxide radical, hydrogen peroxide and nitric oxide. Superoxide radical and hydrogen peroxide are logical since they are the substrate and product of the superoxide dismutase enzymatic reaction, respectively. Nitric oxide radical is also logical since it reacts with superoxide with a rate constant comparable to that of MnSOD. Our work has focused on testing hydrogen peroxide as an effector, since there are enzymes that specifically remove this species. Thus, we have already transfected cytosolic glutathione peroxidase (GPx1) into MnSOD overexpressing cells and demonstrated a modulation of the tumor suppressive effect in all cell lines tested. Surprisingly, in one cancer cell line, GPx1 overexpression caused an inhibition of the tumor suppressive effects of MnSOD [17], while in another cell line GPx1 overexpression actually increased the tumor suppressive effect of MnSOD [19]. A major goal of our future work will be to understand why different results are obtained in different cell lines. Since GPx1 can also remove other hydroperoxides, it is also unclear if its effect is due to hydrogen peroxide or other hydroperoxides. For this reason, we also plan in the future to transfect both peroxisomal and mitochondrial catalase (made by adding the MnSOD presequence) into MnSOD overexpressing cells to determine the effect. Catalase does not effectively act on lipid hydroperoxides, but does remove hydrogen peroxide effectively. Recently, Rodriguez et al. [34] have shown that either peroxisomal or mitochondrial plus peroxisomal catalase can reverse the MnSOD-dependent inhibition of cell growth and plating efficiency seen in a human fibrosarcoma cell line. These results suggest that hydrogen peroxide is involved in the tumor suppressive effects seen with MnSOD and this work with catalase overexpression needs to be reproduced in other transfected cell lines. Very recently, work has been published that supports a strong role for hydrogen peroxide, in the growth suppressive effect of MnSOD [8]. Davis et al. [8] have shown potent growth inhibitory effects of an active site mutant of MnSOD that has increased enzymatic efficiency. His-30 of MnSOD is part of a hydrogen-bonding network in the active site, which may be important for the delivery of protons to regenerate the active Mn(III) enzyme. Davis et al. [8] made a H30 N (substitution of asparagine at His-30) mutant that has greatly increased catalytic efficiency. The authors tried to make stable H30 N overexpressing cell lines by plasmid transfection of HEK 293 cells, but were unable to do so because the selected
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cells would not grow. Over the time period necessary for stable selection, overexpression of the H30 N protein caused significant slowing of cell growth in multiple independent clones and separate transfections, irrespective of the nature of the vector or selection regimen. It was found that the levels of ROS measured by DCF-DA fluorescence were greatly increased in these cells. Since catalase reduced the ROS signal, it was hypothesized that hydrogen peroxide caused the antiproliferative effect of the mutant enzyme. To examine this hypothesis, clonal cell lines that stably overexpressed mitochondrial catalase (mtCAT) were first established; these lines were then doubly transfected with plasmids containing H30 N. As predicted, the inclusion of mtCAT eliminated the quiescent phenotype associated with H30 N transfection and suggested that hydrogen peroxide was responsible for the growth inhibition. The authors also showed the growth inhibitory effect of MnSOD overexpression using retroviral-mediated gene targeting of A549 human lung adenocarcinoma cells. This technique allowed transduction in a much smaller period of time. With this technique, H30 N overexpressing cells were isolated and grew much more slowly than control, vector transduced, or wild type (WT) MnSOD transduced cells. Immunoblot analysis showed similar amounts of immunoreactive protein in WT or H30 N-overexpressing cells, thus excluding the possibility that changes in MnSOD protein levels were responsible for the slower growth of the mutant cells. To test the antitumor effect of H30 N in vivo, A549 cells were transduced with a retroviral vector. Forty-eight hours following transduction, cells were injected subcutaneously into NOD/SCID mice and tumor volume was assessed with time. A great inhibition of tumor growth was observed in H30 N mice compared to those transduced with vector alone or WT MnSOD. The authors conclude that H30 N has a greatly increased growth inhibitory capacity due to its greater production of hydrogen peroxide. This study strongly supports the hypothesis that the growth inhibition caused by MnSOD overexpression is due to the hydrogen peroxide produced.
7. Effect of eNOS on the tumor suppressive effect of MnSOD Andres Melendez has suggested that nitric oxide is also involved in the suppression of cancer cell proliferation by MnSOD [25]. Nitric oxide is known to react with superoxide radical with a rate constant higher that of superoxide with MnSOD; thus, nitric oxide and MnSOD compete in cells for superoxide radical. Dr. Melendez found that MnSOD overexpression enhanced the cytostatic action of three nitric oxide donor compounds. Thus, nitric oxide enhanced the inhibition of cell proliferation caused by MnSOD overexpression. In the Melendez paper, the effect of MnSOD overexpression was lost at high concentrations of nitric oxide donors. Hence, nitric oxide concentrations may determine the results. More work is necessary to examine the possibility of nitric oxide involvement. If neither CAT nor GPx nor NOS affected the tumor
suppressive effect of MnSOD, we could conclude that the sole effector molecule was superoxide radical; the above data does not support this possibility in the few cell lines examined. We have also examined the effect of nitric oxide by transfecting the endothelial nitric oxide synthase gene (eNOS) into a human oral cancer SCC-25 cell line [22,20]. We found that overexpression of eNOS inhibited oral cancer growth in vitro, but in vivo the transfectants grew faster than wild type or vector controls [22]. So different results were obtained in vitro compared to in vivo. One possible explanation for these results is that NOS can under certain conditions produce not only nitric oxide, but also superoxide radical. Different species may have been produced by NOS in in vitro conditions compared to in vivo conditions. In addition, NO• can diffuse out of the transfected tumor cell, so that in vivo, effects on surrounding normal cells are also possible. In fact, increased normal cell proliferation after eNOS transfection appeared to be present in our in vivo model [22]. We also previously transfected eNOS into an MnSOD overexpressing clone [20]. Little effect was seen in vitro, but in vivo eNOS overexpression actually inhibited about 50% the tumor suppressive effect of MnSOD. This work was not published because it was incomplete and must be further studied in the future.
8. Increased anticancer cytotoxicity with BCNU We have found that MnSOD in combination with certain chemicals can have an anticancer effect that causes cell killing in contrast to the non-cytotoxic tumor suppressive effect described above for MnSOD alone. The enzymatic effect of MnSOD protein is to dismute superoxide radical into hydrogen peroxide. If we inhibit hydrogen peroxide removal, then we should kill cancer cells because of direct toxicity or hydrogen peroxide-mediated damage. We have tested this idea in tissue culture using stable plasmid transfected rat glioma cells and found very positive results [48]. The higher the MnSOD levels, the higher the killing we observed in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or buthionine sulfoximine (BSO). BCNU is a clinically used anticancer drug that causes alkylation and also inhibits glutathione reductase (GR). If GR is inhibited, cells cannot remove hydrogen peroxide [48]. BSO is a competitive inhibitor of a glutathione synthesis protein. If BSO is given to cells, then glutathione is depleted and again hydrogen peroxide cannot be removed by the glutathione peroxidase system. As an example of the effectiveness of this combination, with wild type and vector control cells, about 10% of the cells were killed by 50 µM BSO, while 100% of the high MnSOD overexpressing cells were killed by the same dose of BSO [48]. These data again suggests that hydrogen peroxide is a mediator of the tumor suppressive effects of MnSOD. The significance of this observation is that MnSOD overexpression can be used as the basis for a new anticancer combination therapy. We have shown that adenoviral MnSOD when given with BCNU is a power-
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ful antitumor therapy in a mouse xenograft model of human oral cancer [41].
9. Significance The significance of this work is the understanding of the mechanism of the tumor suppressive effect of MnSOD. If we can determine the molecular species responsible for the tumor suppressive effect, then we can maximize this pathway to produce the largest effect on cancer cell growth. We are already developing therapies based on overexpression of MnSOD and the proposed work will help us maximize that therapy. An example of where we could go from this research is that if we determined that hydrogen peroxide caused the tumor suppressive effect of MnSOD, we could develop inhibitors of all the hydrogen peroxide removing molecules in the mitochondria of cancer cells to be given in combination with MnSOD overexpression. We feel that overexpression of MnSOD has real potential in cancer therapy.
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