Lasers Med Sci DOI 10.1007/s10103-012-1114-3
ORIGINAL ARTICLE
Photodynamic therapy for the treatment of induced mammary tumor in rats Isabelle Ferreira & Juliana Ferreira & José Dirceu Vollet-Filho & Lilian T. Moriyama & Vanderlei S. Bagnato & Daisy Maria Favero Salvadori & Noeme S. Rocha
Received: 25 August 2011 / Accepted: 26 April 2012 # Springer-Verlag London Ltd 2012
Abstract The objective of this work was to evaluate photodynamic therapy (PDT) by using a hematoporphyrin derivative as a photosensitizer and light-emitting diodes (LEDs) as light source in induced mammary tumors of Sprague–Dawley (SD) rats. Twenty SD rats with mammary tumors induced by DMBA were used. Animals were divided into four groups: control (G1), PDT only (G2), surgical removal of tumor (G3), and submitted to PDT immediately after surgical removal of tumor (G4). Tumors were measured over 6 weeks. Lesions and surgical were LEDs lighted up (200 J/cm2 dose). The light distribution in vivo study used two additional animals without mammary tumors. In the control group, the average growth of tumor diameter was approximately 0.40 cm/week. While for PDT group, a growth of less than 0.15 cm/week was observed, suggesting significant delay in tumor growth. Therefore, only partial irradiation of the tumors occurred with a reduction in I. Ferreira (*) : N. S. Rocha Faculdade de Medicina Veterinária e Zootecnia, Departamento de Clínica Veterinária, Universidade Estadual Paulista (UNESP), São Paulo, Brazil e-mail:
[email protected] J. Ferreira Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, São Paulo, Brazil J. D. Vollet-Filho : L. T. Moriyama : V. S. Bagnato Instituto de Física de São Carlos, Departamento de Física e Ciência dos Materiais, Laboratório de Biofotônica, Universidade de São Paulo (USP), São Paulo, Brazil D. M. F. Salvadori Faculdade de Medicina, Departamento Patologia, Universidade Estadual Paulista (UNESP), São Paulo, Brazil
development, but without elimination. Animals in G4 had no tumor recurrence during the 12 weeks, after chemical induction, when compared with G3 animals that showed 60 % recurrence rate after 12 weeks of chemical induction. PDT used in the experimental model of mammary tumor as a single therapy was effective in reducing tumor development, so the surgery associated with PDT is a safe and efficient destruction of residual tumor, preventing recurrence of the tumor. Keywords PDT . Adjuvant therapy . Mammary tumors . Comet assay
Introduction Mammary tumors are the most common cancer in women worldwide and represent a remarkably heterogeneous group in terms of morphology and biological behavior. They are also the most representative type of cancer in female dogs [1, 2]. From the epidemiological profile, the mammary tumor apparently results from the interaction of genetic factors, reproductive behavior, hormonal factors, diet, as well as exposure to radiation and chemicals [1]. Despite the advances in diagnosis, in Brazil, this tumor is usually diagnosed in the advanced stage of the disease, when treatment is less effective. The conventional protocols for the treatment of cancer are surgery, chemotherapy, and radiotherapy, used alone or in combination. Except for surgery, the mechanism of action of those therapies on cells is nonselective destruction. Because of this both neoplastic and normal cells are destroyed, thus the side effects of the therapies are undesirable. Since conventional therapies have low efficiency and side effects,
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new techniques are being researched for the treatment of cancer, including mammary tumors. There are some common steps in the development of mammary tumor in women, dogs, and rats [3, 4]. Out of the major groups of cancers currently diagnosed, the mammary tumors appear as frequently in women as in dogs. Rats as an experimental model are the most widely used for studies of mammary carcinogenesis, because they are standardized and comparable to the spontaneous disease in women. When ethically used, experimental models are a valuable alternative to investigate these common points. Mammary tumors are induced by administering the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), which is a polycyclic hydrocarbon that requires metabolic activation [5], to young virgin rats, and is one of the most used experimental systems. The characteristics of this process make this model comparable to spontaneous disease in women. To study mammary tumors in vivo experimental models for comparison with other species, many factors must be taken into account, such as diet, age, hormonal factors, dose, and route of administration [3, 5–12]. Sprague–Dawley (SD) rats are among the most sensitive to carcinogen-induced mammary cancers [8, 13]. An early study identifying the mammary window of susceptibility showed that the peripubescent period of about 45–50 days old is the optimal time for initiating the DMBA model [14, 15], which is a well-known model for the production of tumors that are morphologically heterogeneous and hormone dependent. Photodynamic therapy (PDT) shows considerable promise as a treatment technique for the clinical management of a variety of cancers. The clinical treatment regimen consists of cytotoxicity induction of proliferative cells through the interaction of a photosensitizing agent (PS), light of a proper wavelength, and molecular oxygen [16, 17]. Ideally, PS accumulates mainly in neoplastic cells and is activated by light. This activation transfers the molecules of the photosensitizing agent into excited state molecules [17]. One of the manners by which excited molecules return to the ground state is by transferring the absorbed energy directly to cellular oxygen, generating highly reactive singlet oxygen, which is responsible for cell death and known as type II reaction [17, 18]. Both vascular and direct cell damages contribute to tumor destruction [19–21]. Alternatively, excited molecules can transfer energy to the intermediate molecules, which then react with oxygen to produce free radicals, a type I reaction. In addition, the role of the inflammatory process and the immune system has been studied as regards to PDT [20, 21]. However, for the success of PDT, knowledge of light distribution within biological tissue is vital because this type
of therapy depends on penetration and minimal amount of light in the target tissue [22]. The propagation of light in tissues is influenced by their optical properties [23, 24]. Optical fibers can be used in contact with the surface and also be inserted into the middle of the tissue being investigated in order to measure the local intensity of light [22, 25–28]. PDT has several advantages; the primary is selective tissue destruction. Side effects are reduced, maintaining the aesthetics and with no observed drug resistance. PDT can be used alone or in combination with other therapies. Thus the response of patients undergoing PDT is satisfactory [17–29]. The comet assay is one of several methods of DNA fragmentation detection which uses single-cell gel electrophoresis to detect DNA strand breaks in individual mammalian cells. The comet assay is a relatively simple technique that allows to determine not only the percentage of DNA damage but also the extent of genotoxic damage [30]. Thus, the search for less invasive therapies with reduced side effects has motivated this study and has the purpose of obtaining information on the effectiveness of PDT as a single therapy and adjuvant surgery in chemically induced tumors in rats of SD strain. This analysis was made by cytology, histology, and testing of the comet.
Materials and methods Light absorption To study light distribution in vivo, two Sprague–Dawley rats (animal 1 and animal 2) without mammary tumors were used. The light-emitting diode (LED) device described in the “Photosensitizer” section was used as a light source, with the same irradiation parameters used for the treatment of tumors. The investigation sites included the skin surface, subcutaneous tissue, and fat tissue adjacent to mammary gland. For the light distribution measurements, mammary tissue was used in different locations. For animal 1, thoracic was used, and for animal 2 an inguinal was chosen. The choice of different sites is due to the difference in the amount of adipose tissue present on them, since the inguinal region has more fat accumulation. Animals Twenty young, virgin Sprague–Dawley female rats were used in this study. The animals were bred in vivarium of the medicine school of UNESP—Botucatu. The photosensitizer was maintained in accordance with the guidelines of
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the committee on care and use of laboratory animals of the Brazilian National Research Council (CNPq) and the commission for ethics in research of the School of Veterinary Medicine and Animal Science at UNESP—Botucatu. They were fed with appropriate rat food in pellets, given filtered water, and kept under ideal conditions of temperature, humidity, and light. Photosensitizer Photogem® (Photogem, Moscow, Russia), a hematoporphyrin derivative complex, obtained by Photogem LLC Company in Moscow, Russia was used as photosensitizer. A stock solution of the photosensitizer of 5 mg mL−1 in 20 mM phosphate-buffered solution with 0.9 % NaCl, pH 7.4 (PBS) was stored in the dark at 4°C. This photosensitizer consists of a mixture of monomers, dimers, and oligomers and holds up to eight porphyrin units, linked together by ether or esther. This photosensitizer is produced using original technology of animal and human blood defibrillation; this same photosensitizer came from hematoporphyrin IX which is present in the blood circulation (Fig. 1). Light source The region of the electromagnetic spectrum emission for the PDT light source was chosen by taking into account the absorption bands of the PS and the light penetration into biological tissues. For PDT application, a LED device composed of an array of red LEDs was used. The Physics Institute of São Carlos, University of São Paulo (IFSCUSP) developed this device, which has a wavelength at 635 nm, total emitting power of 1 W, and fluence rate of 185 mW/cm2 [31–33]. The LED device was calibrated in a uniform output each time it is turned on.
Experimental design Mammary tumors were induced by a single dose of 50 mg/ kg of DMBA diluted in soybean oil given by gavage. All animals (between 180 and 230 g; average weight 205 g) received the chemical carcinogen at the age of 50 days. After induction, the animals underwent daily viability inspection, and weekly physical examination, until the tumor development. When neoplasia was identified, we applied the TNM system and performed cytological exam. After that 20 animals were divided into four groups: control (G1), PDT only (G2), underwent surgical removal of tumor (G3), and submitted to PDT immediately after surgical removal of tumor (G4) and studied for 50 days. The control group was clinically monitored only, while the G2 and G4 underwent photodynamic therapy using Photogem® as the photosensitizer (5 mg/kg, IV) [34]. In G2, tumors were irradiated 24 h after PS administration. Irradiation was performed using the LED device with fluence rate 180 mW/cm2 and fluence 200 J/cm2 [32]. For this procedure, the animals were anesthetized with atropine (0.044 mg/kg subcutaneously), then 10 min later an intramuscular injection was administered of xylazine 2 % (20 mg) and ketamine (50 mg) at the dosage of 0.2 ml per 100 g of rat. In G3, the tumors were induced, and after 45 days, the tumors were surgically removed for microscopic analysis. In G4, the animals were irradiated, right after the surgical procedure, 24 h after PS administration. Irradiation was performed using the LED device with fluence rate 180 mW/cm2 and fluence 200 J/cm2 [32]. The animals were submitted to the same procedure as that of G2. At the end of the experiment, the animals were deeply anesthetized with isoflurane and then euthanized by transection of the jugular veins. After that, a complete necropsy was done to verify metastases by clinical TNM. The lesions were analyzed by the routine H&E method. Comet assay
Fig. 1 Chemical structure of the derivative of hematoporphyrin (Photogem)
Alkaline comet assay was used to detect DNA fragmentation of cells. In the control group and immediately after treatment, slide preparation was done by 5 μl of neoplastic cells and PBS free of Ca2+ and Mg2+. This sample was mixed with 100 μl of agarose of low melting point 0.5 %, and distributed on microscope slides precoated with agarose 1.5 % normal melting point, and covered it with a coverslip. After 5 min at 4°C, the coverslips had been removed and the slides were immersed in lysing solution at 4°C for 24 h, in order to dispossess the components of cell membranes. After lysis, slides were washed for 5 min with PBS free of Ca2+ and Mg2+, then were taken to the electrophoresis, immersed in electrophoresis solution, and allowed to rest
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for 20 min for DNA denaturation, 20 min in running. Then the slides were neutralized with neutralizing solution for 15 min, fixed them jump right in 95 % ethanol, dried, and stored until analysis. The sample was stained with ethidium bromide and analysis was performed with a fluorescence microscope at a ×400 magnification. A percentage of the number of cells with DNA damage was generated by counting the number of noplastic cells with comet tail among 100 randomly selected cells per slide. Statistical analysis A repeated measures mixed model (PROC MIXED, SAS Institute, 2009) was used to compare the means of tumoral growth between groups. The interaction between groups and days was significant (P
