Evaluation of the Lobelia inflata Extract in the Histopathological Profile of Melanoma in Experimental Model

Melanoma is a type of skin cancer extremely aggressive affecting more than one
thousand people in the world. Late stages and widely spread malignant melanoma do
not respond to standard therapeutic approaches. Therefore, in this study we induced
the injecting primary tumor with melanoma B16F10 cells in mice and treated the
animals with Lobelia inflata for 22 days to evaluate the antitumor potential of the plant
extract. The animals were monitored, the skin and cutaneous tumor were excised, and
the tissues were prepared for histopathological and morphometric analyses. Regarding
to microscopic aspects, there was a reduction in the angiogenesis process ratified by
the reduction of the number of blood vessels after interaction with the extract and, in
addition, part of the chronic inflammation, reflected by the number of macrophages
decreased after treatment of the mice that received the extract.


Introduction
Cutaneous melanoma is a malignant neoplasm with a multifactorial etiology, characterized by the abnormality of melanocytes that invade the basement membrane. Neoplastic cells use proteins produced by fibroblasts that also release growth factors that melanoma is not able to synthesize. These growth factors can increase the growth capacity and invasion [1]. This is one of the most aggressive neoplasms, predominantly among the white population due to its greater sensitivity to sun exposure [2]. It is a tumor recognized by elevated mortality due to its high metastatic, high aggressiveness and low response to conventional therapies. In spite of corresponding to 4% of the neoplasias, cutaneous melanoma is responsible for 80% of deaths being thus considered a serious public health problem [3]. The earlier the lesions are, the greater the chance of cure, but the risk of death grows in proportion to the increase of the thickness of the tumor [4]. On average, patients who are at late-stage melanoma survive from 6 to 10 months [5]. Distinctly from the disease diagnosed at the initial treatment, which is effectively treated with surgical, the metastatic stage is almost universally fatal, with the response rate to chemotherapy treatment being less than 30% of cases [6].
Despite its seriousness, the available therapeutic for the treatment of this tumor often results in unsatisfactory results. Since advanced melanoma does not respond rapidly towards standard therapies such as chemotherapy, radiotherapy and surgical excision, the search for new anti-melanoma agents is of an urgent need. In this regard, studies have been developed seeking for alternative therapies through the use of medicinal plant-based products for the treatment of melanoma. It is known that Lobelia inflata is a plant belonging to the family Campanulaceae, known as Indian tobacco, popularly widely used because of its emetic, hypnotic, anti-asthmatic, astringent and tobacco substitute properties in alternative therapies. This species is a source of alkaloid substances that motivated studies since its isolation in 1885, when its therapeutic properties was thought to be understood [7]. There are a lot of other substances isolated in the genus Lobelia such as piperidine alkaloids including lobeline, lobelane, lobelanidine, norlobelanine, and lobelanine. Besides, flavonoids, terpenes and triterpenes, saponins, and coumarins have also been identified and probably have pharmacological effects.
Among the cited species of genus, Lobelia inflata is better known and contains more than 20 piperidine alkaloids, being the subject of much research regarding the properties of its extract. On the other hand, another species, Lobelia chinensis, has also become the focus of several studies and explained literature that has known antitumor activities in the treatment of breast and stomach tumors, as well as hemostatic, diuretic and anti-inflammatory characteristics. Some flavonoids are specially found in Lobelia chinensis and are responsible to modulate key cellular enzyme function, acting as anti-cancer, anti-mutagenic, antioxidant and anti-inflammatory substance [8]. However, little is known about the antitumor effect of Lobelia inflata on growth and development of melanomas. It is necessary to discover and understand the probable mechanism of action involved in the interaction between Lobelia inflata and cutaneous melanoma. Thus, upon these considerations, in the present work, we took advantage of B16F10 melanoma cells, which is a well validated model for both primary tumor and we investigated the antitumoral activity of Lobelia inflata during melanoma progression.

Experimental Design
Balb/c mice (n=7/ experimental group) were divided into four groups:

Cell Culture
B16F10 melanoma cells were cultured in culture medium and, once confluent, in the exponential growth phase, were counted and adjusted to the concentration of 10 6 cells / 0.1mL of 0.9% saline.
This model is widely used in research on mice to induce melanoma by the action of a B16 derived retrovirus capable of transforming normal melanocytes into cancer cells [9].

Lobelia inflata Extract
The extract of the Lobelia inflata plant was performed as previously described [10].

Animals Survival and Tumor Growth
Tumor weight and size were monitored and measured every other day for 22 days. Tumors were measured with a Vernier caliper, and their size (in mm 3 ) was calculated, as previously described [11]. Briefly, tumor volume = (length × height × width)/2. For this experiment, mice were monitored for 30 days after tumor injection.
The macroscopic evaluation of the animals was performed daily in relation to tumor growth, according to the ABCD system, observing the possible formation of ulcers in the skin. This rule is relevant for detecting cutaneous melanoma. The letter A characterizes the asymmetry of the lesion, while the letter B means the presence of irregular borders, the letter C suggests color changes and the letter D corresponds to a tumor diameter greater than 6 mm, being all these factors important for determining the prognosis [12].
Mortality was monitored through daily observation over the entire experimental period. Finally, the sacrifice occurred after 22 days of tumor implantation.

Histopathological Evaluation
The tumor arise in the skin was excised and the samples were

Morphometric Analysis
Tumor morphometric analysis was performed using the ISC program, Tucsen Photonics Co., Ltd. in conjunction with a Nikon (Nikon eclipse E200) microscope, coupled to a video camera (Tucsen USB 2.0 H Series) and a computer online. The analysis was evaluated by a researcher blinded to the protocol design.

Statistical Analyzes
The Student's t-test two-tailed was applied for comparison between the groups exposed to the same stress factor (tumor).
Thus, the control groups served as witnesses and were excluded from the analysis because they did not present such a factor common to the categories Melanoma and Melanoma + Lobelia. Data are expressed as mean ± SEM (Standard Error of Mean). Differences between mean values were considered significant when p < 0,05.

Animal Monitoring and Tumor Growth
In order to evaluate the contribution of Lobelia inflata extract against the growth of the melanoma, we used a model of suspension of B16F10 lineage cells in mice from one group that did not receive treatment and in another group that received the Lobelia extract.
Tumor growth was monitored daily in the animals. With respect to the obtained macroscopic results, the Melanoma group was the only one whose animals developed tumor initially, with 57% of the

Reduction of Angiogenesis by Lobelia inflata
Angiogenesis is a central process during cancer development and therefore is considered one of the hallmarks in melanoma.

Analysis of Cell Proliferation by Tumor Mitotic Index and after Treatment with Lobelia inflata
It is known that among the prognostic factors used to classify melanoma aggressiveness, mitotic index is the most accepted and clinically used. When the two groups were compared, it was found that there was no significant difference in the means of the number of mitoses between the untreated group and the one that received the Lobelia inflata treatment, as shown in Figure 3. This suggests that the presence of the extract did not influence the reduction of the number of mitoses in this experimental model (Figure 3).

Analysis of Inflammatory Cells in Melanoma after Treatment with Lobelia inflata
Neutrophils and macrophages were quantified in all animals of both groups (Melanoma and Melanoma + Lobelia). The mean number of neutrophils showed a significant difference between the two groups. As shown in Figure 4, the presence of the extract exerted influence on the neutrophil count, and the group that received it showed a greater dispersion of the data around the mean in relation to the Melanoma group. In this case, the number of neutrophils suggests that acute inflammation was predominant in the group receiving the extract.  The mean number of macrophages showed a significant difference between the two groups. The Melanoma group presented greater variability and higher median value in relation to the other group, which suggests the onset of chronicity of the present inflammatory process, especially in the animals that did not receive the plant extract ( Figure 5). In controls groups 01 (PBS) and 02 (Lobelia), no evident morphological alterations could be seen in the skin.

Discussion
In this study, we used the B16F10 melanoma cells, which is a well validated model for primary tumor. The F10 line of B16 melanoma has been widely used by subcutaneous injection due to its high aggressiveness and also because it has great metastatic potential [9]. It is rationally to infer that Lobelia inflata had influence in the dynamics of the interaction between normal cells and the tumor, as well as in the elements that determine its microenvironment.
It is noteworthy that melanoma presents an early stage of radial growth and its behavior involves less aggressiveness and a later vertical phase, in which the reticular dermis and the subcutaneous tissue are affected, presenting worse prognosis [15]. Therefore, the probable effect exerted by the plant reflects the delayed growth of the tumors in the treated group, besides justifying the smaller dimensions when compared to the untreated animals. Thus, cutaneous melanoma developed in rats that did not receive the extract would present a more aggressive behavior and could be associated with a worse clinical outcome. During all experimental period, only one mouse from the Melanoma group died, which corresponded to 4%, in the total of animals. Angiogenesis is a crucial process during tumor formation and also in the metastatic process, requiring the formation of new vessels so that the cancer cells settle in the target site and maintain a continuous growth.
Invasive capacity is ensured by nutrients, oxygen and by the removal of toxic residues, and thus invasive tumors are a common site of angiogenesis, stimulated by factors such as VEGF (Vascular Endothelial Growth Factor) [16]. Lobelia inflata contains two subfamilies of polyphenols that differ in composition [17]. It is worth mentioning that these compounds manifest wide biological activities, including antibacterial, antioxidant, anti-inflammatory and anticarcinogenic activities [18]. Moreover, angiogenesis is also targeted by polyphenols. These substances have been shown to reduce the migration of endothelial cells and vascular smooth muscle cells, which are essential for the process of blood vessel formation.
Besides, these specific polyphenols decrease the expression of angiogenic factors, among them VEGF [19]. Thus, it is suggested that such a relationship between polyphenols and inhibition of angiogenesis is similar to the mechanism responsible for reducing the number of vessels in the treated group, possibly caused by Lobelia. In the initial inflammatory process, the inflammatory cells produce cytokines, growth factors, prostaglandins and leukotrienes, acting as true chemical agents that favor cell reproduction [20].
Upon the interaction of these elements with their receptors, the tumor develops and progresses, stimulating immune responses [21]. However, the immune system can be overcome by tumor cells, since they have a high replicative power, and also due to mutations that promote the inhibition of tumor suppressor genes, the ability to activate proto-oncogenes and the reduction of apoptosis [22].
Moreover, the higher the tumor growth rate, the more it produces inflammation, which, in turn, promotes the tumor's expansion [23]. From these considerations, it is possible to relate the findings concerning the inflammatory process to the greater melanoma

Conclusion
The present study concluded that Lobelia inflata is a potential therapeutic tool, and its mechanisms of action may in the future mean an effective strategy in the fight against melanoma. The

Funding
The research did not receive any external funding.