Effects of methylene blue-mediated photodynamic therapy on a mouse model of squamous cell carcinoma and normal skin
Introduction
Photodynamic therapy (PDT) is used for the treatment of various types of cancers, precancerous lesions, and dermatological disorders. PDT uses dyes or pigments, referred to as photosensitizers, that absorb visible light and induce or participate in photochemical reactions. The photosensitizer is injected into the target tissue under visible light irradiation of an appropriate wavelength, which results in cell damage. Photochemical reactions of types I and II may occur separately or simultaneously to generate cytotoxic products, such as singlet oxygen (1O2), a highly reactive molecule [1,2].
The photosensitizer methylene blue (MB) can trigger the production of high levels of 1O2 or reactive oxygen species. Skin photosensitivity is a common problem after PDT; however, use of MB reduces the risk of developing this condition because it is hydrophilic, rapidly absorbed, and quickly removed from target tissue. MB also absorbs photons at wavelengths within the therapeutic window (600–800 nm), at which light penetration of the target tissue is greatest [3]. The efficacy of MB-mediated PDT (MB-PDT) in inactivating microorganisms has been demonstrated previously [4,5]. Moreover, in vitro studies have verified the effectiveness of MB as a photosensitizer for the induction of tumor cell death [6,7].
Squamous cell carcinoma (SCC) is a type of non-melanoma skin cancer (NMSC). Its incidence, which is increased by exposure to ultraviolet radiation, is higher than that of melanoma among Caucasians [8]. SCC is associated with a high mortality rate because it is a risk factor for the development of other cancers. It is also associated with various other pathological conditions, such as chronic obstructive pulmonary disease, cardiovascular diseases, acute infections, pneumonia, and immune system dysregulation [9]. PDT has been used for the treatment of NMSC, as well as that of acne, actinic keratosis, and viral warts, and for photorejuvenation, in which its promotion of tissue remodeling yields cosmetic benefits [10].
PDT selectively affects tumor cells without injuring neighboring normal tissues [11], directly and/or indirectly causing cell death by activating an immune response or damaging tumor vasculature [2,[12], [13], [14], [15]]. The tumor microenvironment is critical to the success of PDT, and tumors with a high collagen content respond better to treatment, owing to the vascular damage inflicted by this therapy [16].
In the present work, we evaluated the effects of MB-PDT on normal mouse skin and an experimentally induced mouse model of SCC at two arbitrary time points after treatment (24 h and 15 days). SCC was induced using the chemical carcinogens 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This particular model was chosen due to its similarities to SCC development in humans, with initiation, promotion, and progression stages. Our aim was to investigate the mechanisms involved in tissue damage during MB-PDT. We showed that this treatment was able to reduce tumor size, but did not result in complete remission. PDT also decreased cell proliferation and induced cytokine activation in SCC tissues. Its effects on normal skin were more pronounced, with cell death and tissue damage being increased. Thus, given that the same MB-PDT protocol was employed for the treatment of normal skin and SCC, the tissue microenvironment appears to interfere with treatment responses.
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Animals
Sixty-six female albino mice (Swiss Webster, Unib:SW) were obtained from the breeding colonies of the Multidisciplinary Centre for Biological Research (CEMIB, UNICAMP, Brazil). The experiments were conducted in the Department of Pathology of the College of Veterinary Medicine and Zootechny at the University of São Paulo. The animals, which were 6–7 weeks old and weighed an average of 27 ± 2 g, were divided into separate cages (5 animals/cage) according to their weight. They were kept in
Macroscopic evaluation and tumor size
The tumors induced by DMBA and TPA treatment were highly heterogeneous, including those having developed within the same animal. Treatment response differed between small and large tumors. Small tumors (Fig. 1A) became necrotic (Fig. 1B), whereas larger tumors (Fig. 1C) ulcerated after treatment (Fig. 1D). Mice in the untreated normal skin group (N) exhibited no lesions after hair removal (Fig. 1E). Most mice in the N + PDT + 24 h and N + PDT+15 days treated normal skin groups demonstrated mild
Discussion
In the present study, we investigated the effects of a single MB-PDT session on SCC and normal tissues and the mechanisms involved in the tissue response to this treatment. Our results revealed changes related to the mechanisms of action of PDT, namely, apoptosis, immune system activation, and vascularization. Other variables were also affected by this treatment, including tumor size, cell proliferation, and collagen content.
Tumor size decreased after a single MB-PDT session, consistent with
Conflicts of interest statement
The authors declare no conflicts of interest.
Acknowledgments
The authors thank FAPESP (São Paulo Research Foundation) for the financial support provided (grant numbers: 2009/51336-3, 2010/52675-3, and 2011/19752-7).
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