Cu/Zn superoxide dismutase modulates phenotypic changes in cultured fibroblasts from human skin with chronic radiotherapy damage
Introduction
Superficial radiation-induced fibrosis (RIF), a late effect of therapeutic or accidental high-dose irradiation, is well known in clinical practice. After an asymptomatic cutaneous prefibrotic phase, it is characterized by local inflammation and swelling. With time, this early active RIF undergoes step-wise aggravation and becomes old constituted fibrosis, often clinically associated with superficial atrophy and gradual destruction of cutaneous and subcutaneous normal tissues. After local aggressive physicochemical treatment, or in predisposed patients, the old RIF may sometimes lead to late radiation necrosis [10], [11].
The postulate of the irreversibility of established fibrosis was challenged by clinical observations in patients, in whom it was possible to reduce, significantly and durably, chronic radiotherapy damages using exogenous Cu/Zn superoxide dismutase (SOD) in a liposomal form (LipSOD) [12]. The therapeutic effect was a mean regression of 60% of RIF surface area in two thirds of patients. This result was subsequently reproduced under experimental conditions, in our pig model simulating accidental overexposure [21], where a normalized tissue vascularization and histological signs of muscular regeneration were observed [20]. However, although the role of SOD is the dismutation of superoxide radicals, many questions remained unanswered concerning the mechanisms by which SOD reverses the fibrotic process and permits the replacement of scar tissue, characterized by a dense and hyalinized matrix, poorly vascularized with fewer fibroblasts [7], [9], [33], by normal tissue. Therefore, it was necessary to study these mechanisms in an in vitro human model. In vitro, in monolayer cell culture, some fibrotic cells exhibited an enhanced growth potential, whereas others displayed either the same or a reduced potential as the control fibroblasts. It's the reason why we have developed a human fibroblast protocol from samples of skin treated by radiotherapy 6 months to 9 years before [13]. This culture was possible and reproducible, but limited because of a high rate of spontaneous cell death. The ‘surviving RIF cells’ displayed major signs of exhaustion with a reduced proliferation ability, unchanged tissue inhibitor of metalloproteinases (TIMP) and transforming growth factor beta (TGF-β1) antiproteasic activity. and a reduction of Mn SOD and catalase antioxidant activities [13].
We postulated that the mechanisms of LipSOD action in vivo might be an activation of depleted endogenous antioxidant metabolism, a key regulator of cell proliferation, and an activation of the proteases activities through the down regulation of TIMP and IGF-β1 expression, then the matrix degradation. Most questions concerning the mechanisms governing this in vivo reversion of the fibrotic tissue after treatment with LipSOD are still unanswered. To explore the possible actions of LipSOD on the cellular antioxidant metabolism and the regulation of matrix degradation, we studied the effects of LipSOD on cultured fibroblasts from human old constituted RIF tissue.
Section snippets
Population
Five irradiated patients, whose mean age was 59 years (range: 46–75) and who had been treated for breast cancer (three women) or head and neck cancer (two men), were given 60–70 Gy as postoperative adjuvant irradiation [13]. Surgical skin specimens, free of evolutive cancer were taken 6 and 9 months after irradiation, and 2.5, 5 and 9 years thereafter. The samples were labeled RIF 1 to RIF 5 and were excised as part of programmed surgery which included a curative procedure approved by our
Untreated cells
Control dermis fibroblasts exhibited higher level of endogenous Mn SOD than Cu/Zn activity (Fig. 1A). As previously described [13], endogenous Mn SOD activity was reduced in RIF cells as compared to control dermis fibroblasts, whereas no change was observed for Cu/Zn SOD activity (Fig. 1A). The amount of catalase protein dropped drastically in RIF cells with a 5-fold reduction factor (Fig. 2C).
LipSOD modulation
Treatment of cultures with a LipSOD concentration four times the standard one was very badly
Discussion
In this in vitro study, fibroblasts from human superficial RIF tissue, were modulated by liposomal Cu/Zn SOD in an attempt to understand the mechanisms by which LipSOD leads to the regression of fibrosis in vivo. However, reference studies with non-vectorized SOD and with the liposome vector alone could not be carried out because the available amounts of irradiated human fibrous tissue were insufficient. Consequently, we were unable to test whether cytotoxic properties of high concentrations of
Conclusion
Our observations on radiation-induced fibrosis and its in vivo reversibility have opened up prospects for new treatment, and we are now able to modify the concept of irreversibility of chronic radiotherapy damage. The treatments designed to weaken the fibrotic process could act by modulating cell phenotype rather than by destroying pathologic scars. Although the role of SOD is the dismutation of the superoxide radical, it may also exert many other indirect biological effects which are currently
Acknowledgements
We are grateful to F. Crechet and P. Pinton (LREG) for their technical assistance.
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