Antitumoral Effect of Lobelia Inflata in An Experimental Mouse Model of Melanoma

Antitumoral Effect of Lobelia Inflata in An Experimental Mouse Model of Melanoma. Abstract Background Melanoma is a type of skin cancer derived from melanocytes with high degree of malig nancy and absence of effective therapy, a fact that motivates the search for new treatments. Medicinal plants provide an interesting source of new compounds with anti-inflammatory and antitumoral properties. The Lobelia inflata plant contains a variety of alkaloids, which have been used as respiratory stimulant, antispasmodic, expectorant, antiemetic. Due these facts, we hypothesized that L. inflata may contain compounds with anti-inflammatory and antitumoral activity. Methods: To test this hypothesis, we used an experimental mouse model of melanoma. To evaluate the effects of the L. inflata plant extract, mice were subcutaneously inoculated with B16F10 melanoma cells, and subsequently treated with the extract via oral route every 5 days for one month after tumor inoculation. Tumor growth was monitored every 5 days and after 30 days, the animals were euthanized. Blood and peritoneal fluid were collected for total and dif ferential leukocyte counting, as for quantification of cytokines (TNF-α, IL-6 and IL-1β) and total protein. Results: Administration of the extract reduced inflammatory cell recruitment, edema, and the production of inflammatory cytokines in the peritoneal cavity. Importantly, the plant extract had a significant impact on melanoma growth. Conclusion: These results suggest that L. inflata contains compounds with significant antitumoral activity against melanoma. Highlights Abstract


Background
Melanoma is a type of skin cancer derived from melanocytes with high degree of malignancy and absence of effective therapy, a fact that motivates the search for new treatments. Medicinal plants provide an interesting source of new compounds with anti-inflammatory and antitumoral properties. The Lobelia inflata plant contains a variety of alkaloids, which have been used as respiratory stimulant, antispasmodic, expectorant, antiemetic. Due these facts, we hypothesized that L. inflata may contain compounds with anti-inflammatory and antitumoral activity. Methods: To test this hypothesis, we used an experimental mouse model of melanoma. To evaluate the effects of the L. inflata plant extract, mice were subcutaneously inoculated with B16F10 melanoma cells, and subsequently treated with the extract via oral route every 5 days for one month after tumor inoculation. Tumor growth was monitored every 5 days and after 30 days, the animals were euthanized. Blood and peritoneal fluid were collected for total and differential leukocyte counting, as for quantification of cytokines (TNF-α, IL-6 and IL-1β) and total protein. Results: Administration of the extract reduced inflammatory cell recruitment, edema, and the production of inflammatory cytokines in the peritoneal cavity. Importantly, the plant extract had a significant impact on melanoma growth. Conclusion: These results suggest that L. inflata contains compounds with significant antitumoral activity against melanoma.

Introduction
Inflammation is a process involved in the pathogenesis and progression of several diseases [1]. It is a physiological response that protects against pathogenic microorganisms but may result in tissue damage. The inflammatory reaction aims to restore the homeostasis affected by sterile injury or infection [1]. Recent process. Currently, a causal relationship between inflammation, innate immunity and cancer is widely accepted, and many of the molecular and cellular mechanisms accounting for this relationship are not well understood [2,3]. In a tumor microenvironment, several pro inflammatory mediators participate of a complex signaling process that causes changes in the niche (external microenvironment), changes in the tissue architecture (cells and extracellular matrix), nutritional support and successive mutations in different genes or groups of genes allowing tumor cells to develop [4]. The neoplastic process associated with inflammation includes leukocyte infiltration, production of cytokines such as TNF and IL-2; chemokines such as CCL 2 and CXCL 8; tissue remodeling and neo-angiogenesis [5,6].
Many leukocytes exert anti-proliferative and cytotoxic activities, partially resulting from their ability to secrete reactive nitrogen and hydrogen species (nitric oxide, peroxynitrite, hydrogen peroxide, superoxide) and proinflammatory cytokines (TNF, IL-1, IL-6) [7][8][9][10]. Melanoma is a type of skin cancer derived from melanocytes (melanin-producing cells). The development of melanoma is not fully understood and has been associated to genetic susceptibility and environmental factors such as the destruction of the ozone layer and / or high exposure to ultraviolet rays [11]. Treatments of neoplasms aim to control the tumor microenvironment and eradicate tumor cells. Treatment modalities include surgical procedures, radiotherapy and chemotherapy, which can be used alone or in combination [2,12]. Besides the side effects and the high cost of conventional therapies, they may not be effective.
Because melanoma exhibits a high degree of malignancy, presents increasing incidence and lacks an effective therapy, the scientific community has been devoted to develop new therapeutics with greater effectiveness and less side effects [13]. Alternatives have been proposed, such as biological therapy that takes into account host defense mechanisms and result in a better understanding of the basic antitumor defense mechanisms [12].

Medicinal plants represent a remarkable source of compounds
for the treatment of various human diseases. They are widely explored in modern medicine due to their large range of chemical and biological diversity [14]. The main advantages of using herbal medicines are the low cost, affordability and usually fewer side effects. Studies performed with medicinal plants are very important for confirming their safety and efficacy [15]. The urgent need for new therapeutic agents with greater efficacy and fewer side effects has drawn a great attention to medicinal plants with antiinflammatory and antitumoral properties [13]. Recently, we have demonstrated that Arctium lappa extract has a potent antitumoral activity in vivo [16]. Lobelia inflata is an annual or biannual herbaceous plant belonging to the Campanulaceae family, native of North America, from southeastern Canada (Nova Scotia to Ontario), reaching some states of the United States of America [17]. Popularly, Lobelia is a medicinal plant known as Indian-tobacco, emetic-herb and asthma-herb. The genus also includes the medicinal species L. cardinalis, L. sifilitica and L. chinensis. The base is composed of piperidine alkaloids (lobeline, isolobelin), as well as lobelic acid, chelidonic acid, glycosides, essential oils, resins and fats [18].
Lobelia has a long history of use on respiratory diseases, including asthma, bronchitis, pneumonia, and post-stroke. In homeopathy, is used isolated or in combination with herbs against smoking, muscle relaxation, nausea, vomiting, skin infections (bites, bruises and ringworm), and have properties that relax tissues and reduce spasms [18,19].
Lobelia Chinensis Lour (LCL) is a popular herb that has been used in Chinese medicine for the treatment of lung cancer, inflammation and snakebite [19]. Recent studies have shown that L. chinensis has anti-inflammatory properties, which reduces NF-κB activity and consequently inhibits the expression of Inducible Nitric Oxide Synthase (INOS), Cyclooxygenase-2 (COX-2), Tumor Necrosis Factor-α (TNF-α), Interleukin (IL) 6), both in vitro and in vivo [20]. In addition, isolated components of the Lobelia plant present antioxidant action in vitro, and hepatoprotective effects in vivo [21]. Because of the anti-inflammatory effects of Lobelia, we hypothesized that it also presents antitumoral activity.
Here, we analyzed the potential of the L. Inflata Hydroalcoholic Extract (Lihe) to protect against development of melanoma in an experimental mouse model inoculated with the B16F10 cell line.
We observed that it affected the number of circulating leukocytes and suppressed cellular recruitment into the peritoneal cavity.
In addition, Lihe inhibited the production of pro-inflammatory cytokines and significantly reduced tumor growth. Collectively, these results indicate that L. inflata should be further explored for the development of new antitumoral drugs.

Preparation of Lobelia Inflata Hydroalcoholic Extract
The extract of L. inflata was obtained from 100 g of ground and dry bark, dissolved in 1 L of 70% ethanol (JT Baker, Belo Horizonte, Brazil). A dark bottle conditioned at room temperature was used for 72 hours with daily shaking, and the extract was then filtered on filter paper. The filtrate was dried using a rotary evaporator Animals were sacrificed after 30 days of tumor injection, and blood and peritoneal fluid were collected from these animals for total and differential cell count and for quantification of inflammatory mediators. The experiments were performed twice.

Tumor Evaluation
Tumor growth and body weight were monitored every 5 days after implantation of B16F10 melanoma cells. The volume of the tumor mass was calculated according to the formula: tumor volume = (length x height x width) / 2 and its dimensions (mm) were calculated with the aid of a pachymeter [22].

Leukocyte Counting in The Peritoneal Cavity and in the Peripheral Blood
Blood samples were obtained from the mice previously anesthetized with ketamine/xilasin by the retro-orbital plexus with the aid of the Pasteur pipette containing heparin. Next, the animals were euthanatized in a CO 2 chamber and 3 mL of PBS was added into the abdominal cavity, which was gently massaged for 1 min.
The peritoneal fluid was collected using a syringe with a needle

Statistical Analysis
Statistical analyses were performed using GraphPad software (San Diego, CA, USA). Data were expressed as mean ± Standard Error of Mean (SEM). The differences between any two groups were evaluated using two-tailed Student's t-test. For comparison of multiple groups, we performed one-way Analysis of Variance    *Tumor group versus tumor + Lihe group. These differences were considered significant with p < 0.05 according to two-tailed Student's t-test.

Tumor-Group
Tumor growth was monitored every 5 days for 30 days after implantation of B16F10 melanoma cells. Lihe treatment significantly decreased the size of the tumor when compared to the group of mice that received only vehicle (Figure 6a and 6b). *Control group versus Lihe group; #tumor group versus tumor + Lihe group. These differences were considered significant with p < 0.05 according to two-tailed Student's t-test. *Tumor group versus tumor + Lihe group. These differences were considered significant with p < 0.05 according to two-tailed Student's t-test . *Tumor group versus tumor + Lihe group. These differences were considered significant with p < 0.05 according to ANOVA with Tukey's post-test.

Discussion
In this study we analyzed the inflammatory process induced by the B16F10 melanoma tumor cells in mice and how this was affected by the oral administration of the Lihe. Inflammation is a critical process that can initiate and/or propagate carcinogenesis.
Tumor cells are capable of producing factors that attract leukocytes to the tumoral environment. In addition to localizing within tumor aggregates, immune cells are frequently present within the stromal microenvironment that sustains tumors [23]. Neutrophils play a significant role during this process [1]. Ours results showed that the recruitment of neutrophils was significantly reduced by Metallo Proteinases (MMPs) [24].
The extravasation of proteins occurs due to the increase in vascular permeability, which is typical of the inflammatory process caused by the tumoral activity [1,16,23]. Our results showed a reduction in protein levels in all the animals treated with the extract, providing further evidence for a reduction of the inflammatory process during tumoral activity. Cytokines are secreted or membrane-bound proteins that regulate the growth, differentiation and activation of immune system [25]. The cellular alterations that give rise to cancer provoke changes in local cytokine expression. These perturbations stimulate immune-cell infiltrates, which, in turn, release additional cytokines that act in an autocrine or paracrine manner. These host reactions to cellular stress can impact several stages of cancer formation and progression [26].
TNF-α is a well-known pro-inflammatory cytokine and a major inducer of NF-κB transcription factor, a key regulator of oncogenesis [27,28]. Activation of NF-κB promotes cellular proliferation and inhibits apoptosis favoring cancer development [29]. Angiogenesis is important step to tumor progression and invasion [30,31]. and transforming growth factor α, and by decreased expression of angiogenic inhibitors, such as interferon-α/β, platelet factor-4, thrombospondins and angiostatin [32]. Melanoma cells produce multiple cytokines and growth factors, including transforming growth factor β, Acidic Fibroblast Growth Factor, AFGF, plateletderived growth factor, IL-8, TNF-α, IL-1, IL-6, VEGF and interferons, some of which have potent angiogenic activity and consequently potentiate neoplastic progression [26,33]. Ours results showed that levels of TNF-α, IL-1 and IL-6 were significantly reduced by the administration of the plant extract on mice with tumor cells. Since these cytokines have important roles in tumor progression and invasion, this result represents an important anti-inflammatory and anti-tumoral of our study plant. The main components of our study plant, Lobelia inflata are the alkaloids [34]. Many alkaloids with anti-inflammatory properties have been described.
For example, Bukitingina, an alkaloid obtained from Sapium baccatum (Euphorbiaceae), showed anti-inflammatory potential in carrageenan-induced paw edema, and decreased levels of PGE2 and leukocyte infiltration in the carrageenan-induced pleurisy model [35,36]; Berberine, another alkaloid found in species belonging to the genus Berberies (Berberidaceae) presents promising antiinflammatory activity by reducing both paw edema and vascular permeability in inflammatory models [36,37]; Theacrine is an alkaloid obtained from Camelia kucha, a plant endemic to China, that presents pharmacological activity on the nervous system such as sedation and hypnosis, besides a potential anti-inflammatory activity [38].
Many plant alkaloids also have proven antitumoral activity with several mechanisms of action such as: Catharanthus roseus (L.) G. Don, also known such as Vinca, which has compounds that are capable of stimulating depolarization of the microtubules [39].
Paclitaxel, a polyhydroxylated triterpene obtained from Taxus brevifolia, a Pacific tree, which obtained positive results against a wide variety of tumors at nano-molar concentrations such as the breast and ovarian tumor due to its ability to inhibit tubulin depolarization [40,41]; Camptothecin is an alkaloid extracted from Camptotheca acuminata Decne (Cornaceae), with topoisomerase I inhibition activity and has been approved for the treatment of colon cancer, and later to the lung and ovary, among others [42].

Conclusion
In conclusion, administration of the hydroalcoholic extract of the L. inflata plant reduced the recruitment of inflammatory cells, edema and inflammatory cytokines (IL-1, IL-6 and TNF-α) in the peritoneal cavity and had a striking impact on tumoral growth.