Histopathological Analysis of Arctium Lappa Extract in the Treatment of Cutaneous Melanoma in C57BL/6 Mice

Souza S M1, Araújo M F1, Silva A A2, Moreira M R A3, Zoccal K F1 and Tefé Silva C1* 1Centro Universitário Barão de Mauá, Ribeirão Preto, Brazil 2Department of Physiology, University of Mississipi Medical Center, Jackson, MS, United States of America 3Department of Clinical, Toxicological and Bromatological Analyzes. Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (FCFRP-USP), Ribeirão Preto, Brazil

in melanoma) [9], inactivation of tumor suppressing genes (e.g., cyclin-dependent kinase inhibitor 2A (CDKN2A) (p16), a gene that is mutated in autosomal dominant familial melanomas [10], and loss of PTEN (phosphatase and tensin homolog) gene. In addition, antiapoptotic signals are activated and neoplasic cells develop the capacity to undergo metastasis. Recently, other processes have been implicated in the development of cancer including immune system evasion, genomic instability, and alterations in cellular metabolism.
Thus, due to melanoma's significant impact on public health many studies have looked for more effective alternatives to treat melanoma. The Arctium lappa, popularly known as burdock, is a plant from the Asteraceae family of European origin [11]. Due to the presence of lignans arctigenin and arctinin, which have anticarcinogenic and anti-tumorigenic properties, extracts from A. lappa seeds 11 have been suggested as potential alternative against cancer. Lignans have been reported to increase sensitivity of tumor cells to chemotherapeutic drugs [12], and to exert cytotoxic effects inducing apoptosis of tumor cells by reducing cells' ability to utilize glucose with consequent impairment of energy production [5,13,14]. Previous reports also suggest anti-cancer properties of A. lappa in prostatic hyperplasia, leukemia and pancreatic cancer [15][16][17]. In face of A. lappa potential antitumorigenic activity and the difficulties in developing better anti-melanoma therapies, in the present study we examined the effects of burdock extract as a novel alternative therapy to attenuate development and progression of experimentally induced melanoma in C57BL/6 mice. For this purpose, we took advantage of B16F10 melanoma cells, are a well-validated model for primary tumor, to induce subcutaneous tumors. We found that chronic treatment with A. lappa extract significantly reduced tumorigenesis, angiogenesis and mitotic index of cutaneous melanoma in C57BL/6 mice.

Animal Care
Female C57BL/6 mice (6-8 weeks old) were maintained at our animal facility at 25 o C, with a 12 h/12 h light/dark cycle, and provided with free access to food and water. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee on Research and Animal Experimentation.

Preparation of Arctium Lappa Extract
A. lappa plant extract was obtained from 100 g of ground dry bark dissolved in 1 liter of 70% ethanol by maceration. A dark flask conditioned to room temperature was used for 72 h, with daily shaking, and was then filtered on filter paper. The filtrate was

Determination of Arctium Lappa Toxicity
To evaluate the toxicity of the A. lappa extract, we inoculated test method-TG 423 423. Each group was inoculated every 5 days for a total of 25 days and the animal's well-being and survival rate were recorded. The following parameters were observed daily: changes in skin, hair, eyes, mucous membranes, respiration, and central nervous system (tremors, convulsion and sedation) and autonomic nervous system abnormalities (tearing, salivation). After 25 days of the first inoculation, the animals were euthanatized in a CO 2 chamber and total and differential counts of peritoneal lavage and blood were examined for the occurrence of an inflammatory reaction. After analyzing the results, the dose of 5 mg/kg was chosen based on higher dose with lower side-effects.

Tumor Implantation
Primary tumor studies were performed using B16F10 melanoma cells. Tumors were induced by injecting approximately 300,000 cells in 100 μL of PBS subcutaneously into the dorsal superior region of mice weighing ~25 g.

Experimental Design
Animals were divided in four groups: 1) Control Group -

Histopathological Analysis
Tumor and adjacent tissues were collected from all animals and analyzed for skin ulcer formation and tumor size. Lungs, liver, skin and heart were also collected and processed for histopathological analysis. The tissues were immersed in 10% formaldehyde and embedded in paraffin. Sections of 5 µm thick were cut and stained with hematoxilin and eosin (H&E).

Morphometric Analysis
Morphometric analysis was performed using an Aristoplan microscope (Leitz, Germany) coupled to a color camera (Leica DFC280, Heerbrugg, Switzerland) and a computer. Mitoses and number of vessels were counted in 15 randomly noncoincident fields at a magnification of ×400.

Statistical Analysis
Data are expressed as mean ± SEM. Differences between groups were compared using one-way analysis of variance (ANOVA) and the Newman-Keuls post-hoc test. For differences between two groups, the Student t test was used. A 5% level of significance was chosen to denote significant differences between the means. Results were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA).

Tumorigenesis
T+PBS group presented a larger melanoma growth rate than the T+Tr group (Figure 1). We observed that tumor growth in the T+Tr groups was markedly attenuated compared to tumor growth in T+PBS group (Figure 1) We evaluated histological slices from epidermis, dermis and hypodermis.

Angiogenesis
Angiogenesis is an important process for the supply of oxygen and nutrients to growing tumors. Fast-multiplying cancer cells require greater demand for oxygen and nutrients than other cells of the body. In the present study, we found that treatment with A. lappa reduced angiogenesis in the tumors of T+Tr mice when compared to T+PBS group (Figure 2).

Cellular Proliferation
The mitotic index is a prognostic factor used to classify tumor aggressiveness. We found reduced mitotic cells in tumors from T+Tr mice compared to tumors from T+PBS mice (Figure 3

Discussion
In this study, we examined the potential therapeutic action of A. lappa extract against malignant melanoma in mice. A. lappa extract has been demonstrated to exert protection against other cancer-associated pathologies [13,15,16,17], but its effectiveness against melanoma tumors in vivo is still unclear. We found that A.

. Previous studies demonstrated that
A. lappa regulates immune cell migration and activation, which correlates with favorable outcomes in mouse models of acute inflammation and melanoma progression. These data showed that tumor implantation induced significant accumulation of total leukocytes and neutrophils in the peritoneal cavity, which was suppressed by A. lappa treatment [18]. Arctigenin, one of the lignans extracted from A. lappa, has cytotoxic actions that appear to be specific for cancerous cells, inhibiting their growth and consequently promoting apoptosis, while sparing normal noncancerous cells [13]. Awale et al. [17]. demonstrated that arctigenin has preferential cytotoxicity against nutrient deficient cancer cells due to arctigenin's ability to eliminate the inherent resistance of these cells to energy deprivation. The authors investigated the potential mechanism of action of arctigenin and demonstrated that the compound blocked the activation of Akt, which plays an important role in growth and development of cancer cells [17].
Here we demonstrated that A. lappa extract reduced angiogenesis, thus decreasing oxygen and nutrient delivery for melanocytic tumor cells and reducing their ability to maintain high mitotic rate. This finding highlights a novel potentially beneficial effect of A. lappa extract in melanoma cancer and suggests that the mechanism of action may be related to nutrient deprivation of tumorigenic cells Gu et al. [14]. evaluated the citotoxicity of arctigenin in A549 cancer cells and demonstrated that arctigenin promoted the death of tumor cells undergoing glucose deficiency by inhibiting mitochondrial respiration and raising reactive oxygen species concentration in these cells [14]. Additional studies are needed to test whether a similar mechanism is responsible for the anti-tumorigenic actions of A. lappa extract observed in the present study Huang et al. [19]. Also reported a decrease of cancer cells in a prostate cancer model by the action of arctinin, another lignan.
Arctinin has been shown to promote detachment of cancer cells in culture dishes [19]. The mechanism for cellular detachment process by arctinin appears to involve increased expression of MUC- Cutaneous melanoma is a tumor famous for its ability to metastasize. About 30% of patients with cutaneous melanoma will present spreading of tumorigenic cells in other tissues [21], mainly hematogenic tissues, but also for lymphatic road and for proximity. This inhibition facilitates the process of stromal invasion and invasion of blood vessels, which are necessary steps for metastasis.
Heparanase is an enzyme that acts both on the cell surface and in the extracellular matrix to degrade heparan sulfate molecules, the main constituent of the endothelial cell layer. The degradation of this component facilitates the penetration of tumor cells into blood vessels and is important for metastasis Pereira et al. [23].
Highlighted the important relationship of MMPs in the development of squamous cell carcinoma, alluding to the possibility of using MMPs inhibiting agents that could indirectly act as antimetastatic drugs in combating not only epidermoid carcinoma, but perhaps melanoma as well. In the present study, metastases were not observed in any of the organs and tissues analyzed, including heart, lung, skin and liver. However, we did not perform molecular examination to specifically determine if A. lappa extract protects against melanoma metastasis.

Conclusion
Our results suggest that Arctium lappa may represent a potential option as novel therapy for cutaneous melanoma cancer treatment.
Our findings demonstrate a beneficial effect of A. lappa extract to reduce melanoma tumor growth, which was associated with reduced angiogenesis and mitotic rate. Studies have showed that arctigenin and other lignans extracted from A. lappa may represent a therapeutic strategy with minimum adverse effects on normal tissues. However, larger, more comprehensive studies are needed to better elucidate the tumorigenic suppressant mechanisms of plant extracts in the treatment of cutaneous melanoma.

Funding
The research was funded by Centro Universitário Barão de Mauá›s intramural faculty grant.