INTRODUCTION: Cancer can be defined as a condition in which malignant cells multiply uncontrollably and invasively, supported by an environment rich in nutrients, growth factors and cells, which promote tumor growth [1]. Its common treatments are surgery, radiation and chemotherapy and they are not capable of differentiating tumor or healthy cells [2, 3]. Drug repurposing is a strategy where existing drugs undergo additional studies to treat different diseases. This approach offers benefits by speeding up the development process for both the pharmaceutical industry and patients [4]. The indicated method is advantageous for cancer treatment due to its utilization of well-established medications, ensuring a safety profile during treatment [5]. Nitro compounds display a vast diversity of pharmacological properties, such as antineoplastic, antibiotic, antihypertensive, among others [6]. Nitrofurantoin is a synthetic molecule obtained from furan, nitro group and hydantoin used as an antibiotic for the treatment of urinary tract infections [7]. Old experiments made by Bulbul et al. [8] showed an antitumoral effect using nitrofurantoin. However, even after almost four decades since this study, there is still a scarcity of research regarding the anticancer properties of this compound. AIMS: This study aimed to evaluate the in vitro and in vivo anticancer activity of nitrofurantoin, analyzing its drug repositioning potential for cancer treatment. METHODS: The nitrofurantoin used for in vitro assays was acquired by Sigma-Aldrich, while the cancer cell lines HL-60 (human promyelocytic leukemia), HT-29 (human colon carcinoma), MCF-7 (human flowing carcinoma) and NCI-H292 (human pulmonary mucoepidermoid carcinoma) were obtained from the Bank of cells of Rio de Janeiro, Brazil, and grown in Cell Culture Laboratory, Department of Antibiotics, Federal University of Pernambuco (UFPE). For the cytotoxicity assay, cells were added to 96-well plates at a concentration of 2x105 cell/mL for adhered lines and 0.3x106 cells/mL for suspended cell lines and a dilution of 0.048 µg/mL to 25 µg/mL, with doxorubicin as a standard control. After 72 h, the supernatant was removed, and was added 25 µL of MTT solution (5 mg/mL) (Sigma-Aldrich®). The plates were left in an oven and, after that period, 100 µL of DMSO was added to dissolve crystals formasan. The absorbance was measured in a microplate reader at a wavelength of 540 nm [9, 10]. For the determination of cell viability, tumor cells were assessed by the exclusion test using trypan blue vital dye (Sigma) 0,4% p/v, in PBS [11], in which HL-60 cell line (0.3x10? cells/mL) was incubated with nitrofurantoin or doxorubicin (0.3 mg/mL), the standard control, and tested inverted microscope. It was removed 90 µL from the suspension and added 10 µL of trypan blue. The cell differentiation was counted in a Neubauer chamber [12]. For the morphological analysis, HL-60 cell line, at a concentration of 0.3x10? cells/mL, was incubated for 24 h with doxorubicin (0.3 µg/mL) for positive control, DMSO (1.6%) for negative control or nitrofurantoin (5 or 10 µg/mL). Slides were prepared in cytospin and, after cell adhesion to the blade, it was stained with hematoxylin/eosin. The recording changes were made by photography. For in vivo testing, which was authorized by the Research Ethics Committee of UFPE under number 23076.037858/2012-93. Swiss albino mice were divided into groups of 08 per cage, and the Ehrlich carcinoma cells (murine mammary adenocarcinoma) were obtained from Bioassays Laboratory, Department of Antibiotics, UFPE. For the determination of hemolytic activity, blood was collected from the Swiss mice by cardiac puncture. Erythrocytes were washed with saline and centrifuged three times in each procedure. After the final wash, suspension of erythrocytes (SE) 2% were plated on a 96-well plate. It was added 100 µL saline for negative control, 50 µL saline + 50 µL for white, 80 µL saline + 20 µL Triton X - 100 1% for positive control, and 100 µL saline + 100 µL nitrofurantoin (from 7.81 to 1000 µg/mL) diluted in 10% DMSO. 100 µL of SE was added to each well and the plate was incubated for 1 h. The optical supernatant was taken in an automatic plate reader [13, 14]. For the evaluation of the in vivo antitumor activity, Ehrlich carcinoma tumor cells were introduced subcutaneously in the axillary region in the recipient mice (0.2 mL of a suspension at a concentration of 5x10? cells/mL, with treatment initiated 24 h after implantation. Nitrofurantoin was tested at doses of 20, 60 and 120 mg/kg, all orally once daily for seven days. The negative group received saline and the positive group received doxorubicin (1.5 mg/kg). On day nine, before euthanasia, the animals were anesthetized with xylazine/ketamine and the blood by cardiac puncture was collected, which total count of erythrocytes, leukocytes, hemoglobin concentration, index hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MHC) and mean corpuscular hemoglobin concentration (MCHC) parameters were evaluated. After blood was collected, the animals were euthanized for resection of the tumor blood collection and removal of the kidney, spleen and liver. The experiments were analyzed by their means and standard errors by variance (ANOVA) followed by Newman-KEuls post-test (p < 0.05) using GraphPad Prism version 5.0. RESULTS AND DISCUSSION: The cytotoxicity analysis showed that nitrofurantoin was more cytotoxic against NCI-H292 cancer cell line (IC50 = 0.9 µg/mL), also showing good cytotoxic effect on the others tumor cell lines (MCF-7 = 3.3 µg/mL; NCI-H292 = 0,8 µg/mL; HL-60 = 1,3 µg/mL). Based on the United States National Cancer Institute (NCI) criteria, the IC50 ? 4.0 µg/mL represents a promising cytotoxicity for pure compounds, classifying nitrofurantoin as a promising candidate for continue anticancer evaluation [15, 16]. Corroborating this, nitrofurantoin, at a concentration of 10 µg/mL, reduced in 62.1% compared to 67.9% of doxorubicin reduction of the number of viable cells. Nonetheless, HL-60 was chosen to evaluate the cytotoxic activity of nitrofurantoin because of its capacity of growing in suspension and its remarkable sensitivity in front of the nitrofurantoin. The tested compound, at a concentration of 10 µg/mL, modified nitrofurantoin-treated HL-60 cells morphology, which showed nuclear vacuoles, pyknosis, cell contraction areas, nuclear fragmentation and a marked decrease in the number of cells. Pyknosis is a condition that occurs when a cell undergoes necrosis, a process in which, among other factors, toxic cellular content is released into the surrounding extracellular environment, leading to local inflammation and death of adjacent cells [17]. In addition, nitrofurantoin was not capable of lysing erythrocyte membranes, which represents a selectivity of cytotoxicity demonstrated by nitrofurantoin in damaging tumor cells (EC50 > 1000 µg/mL) [18]. The test compounds at all doses decreased tumor growth significantly compared to the control group (54.4% to 20 mg/kg; 53.8% to 60 mg/kg and 67.4% to 120 mg/kg). The treatment of nitrofurantoin, at all doses, do not have any substantial changes in the hematological parameters. The treatment of all nitrofurantoin doses reduced the contents of liver and kidney compared to the saline group (negative control). CONCLUSION: Nitrofurantoin showed a more pronounced cytotoxic effect against the NCI-H292 cell line. In the cell viability assay, there was a substantial reduction in viable cells. Morphological analysis of nitrofurantoin-treated HL-60 cells showed characteristics of apoptosis. In vivo antitumor test showed a tumor growth inhibition at all doses that were tested, also showing no toxic effects on hematological parameters studies. Therefore, this study showed the nitrofurantoin antitumor activity. ACKNOWLEDGEMENT: I would like to thank CNPq and Federal University of Pernambuco, which provided me with financial conditions to keep doing research. Moreover, I would also like to thank my laboratory colleagues and my advisor, who allowed me to be part of the laboratory.
REFERENCES
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Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
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