1. Introduction
Cancer is a generic term that refers to a set of diseases characterized by the presence of cells in continuous proliferation with invasion and metastasis properties [
1]. It is one of the most common causes of high morbidity and mortality [
2] which is an important public health problem worldwide [
3]. Tumor cells share several characteristics, including unregulated proliferation, angiogenesis induction, the ability to escape from immunological detection, and tumor-promoting inflammation [
4].
Tumor angiogenesis is one of the markers of tumor progression [
5]. It is necessary for the adequate supply of oxygen and nutrients, besides favoring the migration of tumor cells over great distances, leading to metastasis [
6]. Thus, angiogenesis represents an important therapeutic target against cancer [
7].
The tumor microenvironment is the site of tumor development, which includes not only neoplastic cells, but also stromal cells, such as vascular and lymphatic endothelial cells, pericytes, fibroblasts, myofibroblasts, and various bone marrow-derived cells, such as macrophages and neutrophils [
8]. In carcinogenesis, the microenvironment surrounding the tumor provides signs of tumor suppression, to maintain the tissue homeostasis architecture essentially controlled. Nevertheless, once tissue homeostasis is lost, this altered microenvironment becomes a potent tumor promoter. Then, once the tumor is formed, it initiates complex inflammatory and immune responses, in which inflammatory cells are recruited in response to signals from that microenvironment [
9].
Tumor cells interfere in both innate and adaptive immunity, and induce macrophages and neutrophils to a type 2 differentiation state, in addition to a change in the response profile of Th1 to Th2 lymphocytes [
10]. These cells are able to interfere with each step of the antitumor inflammatory response by secreting mediators that block the function of immune effector cells and reprogram these cells to a regulatory profile [
11]. M2 macrophages and Th2 lymphocytes together lead to immunosuppression, angiogenesis and tissue remodeling associated with the release of a set of cytokines, such as IL-4 and IL-10 [
12]. These collaborative interactions between neoplastic cells and their stroma aggregate into ectopic structures, which are chronically proliferative and often disseminative [
8].
On the other hand, reactive oxygen species (ROS) production by neutrophils and macrophages as a mechanism for tumor cell destruction is already well established. The disproportionate increase of ROS in tumor cells can lead to cell cycle arrest, apoptosis and/or senescence. Furthermore, activated macrophages also generate nitric oxide (NO) that reacts with superoxide to produce peroxynitrite radicals, contributing to tumor cell apoptosis [
13,
14,
15]. Actually, NO has dichotomous effects, modulating different events related to cancer including angiogenesis, apoptosis, cell cycle, invasion and metastasis [
16].
Non-clinical and clinical research using a variety of cancer therapies continues to grow worldwide with the prospect of new drug discovery, or to reduce toxicity and the development of resistance related to current drugs. In this context, natural products continue to play an important role as prototypes to the synthesis of new anticancer drugs [
17,
18,
19].
The piperine alkaloid amide (1-piperoylpiperidine) was originally isolated from fruits of black pepper (
Piper nigrum Linn) and long pepper (
Piper longum Linn) [
20]. Literature data have shown anti-tumor activity in vitro [
21,
22] and in vivo [
23] for piperine. However, piperine and analogues have high toxicity in rodents, characterized mainly by hepatotoxicity [
24].
In this study, we investigated the toxicological and antitumor effects of a novel piperine analogue, N-(p-nitrophenyl)acetamide piperinoate (HE-02), on the Ehrlich ascites carcinoma model and its mechanism of antitumor action, by evaluating angiogenesis and the tumor microenvironment.
3. Discussion
Natural products continue to serve as an important source of anticancer drugs which are used as prototypes for the synthesis of more active and less toxic drugs. In this sense, several piperine analogues have been studied for antitumor and toxicity effects. In the present study, we used piperine as a prototype for the synthesis of a novel analogue, which was tested for toxicity and in vivo antitumor activity.
The cytotoxicity assay in mouse erythrocytes allows us to evaluate the potential of a drug to cause damage to the cell’s plasma membrane, pore formation or the measurement of cell permeability [
25]. In addition, erythrocytes are known to be targets of antineoplastic drugs [
26]. Therefore, the test represents an important model for evaluating the cytotoxicity of new antitumor drug candidates. This study indicated that HE-02 did not induce damage in the cell membrane, producing low cytotoxicity in erythrocytes of mice. Similarly, piperine was not able to induce hemolysis up to 200 μg/mL [
24]. On RAW 264.7 cells, HE-02 induced cytotoxicity from the lowest concentration tested (10 μg/mL), indicating a moderate cytotoxic effect. Nevertheless, until 1000 μg/mL, HE-02 reduced less than 50% of cell viability. For piperine, literature data demonstrate that this substance did not induce cytotoxicity in RAW 264.7 cells at concentrations up to 100 μg/mL [
27].
In vivo studies with HE-02 started with the acute preclinical toxicity test in mice, with the objective of determining safe doses to be used in pharmacological tests. Furthermore, a psychopharmacological screening was performed with the objective of qualitatively detecting some important actions of HE-02 on the central nervous system (CNS) and autonomic nervous system (ANS) [
28]. Considering the LD
50 around 2000 mg/kg, as well as the fact that the only observed effect, diarrhea, disappeared after 4 h of treatment with the highest dose tested (2000 mg/kg), it can be inferred that HE-02 has low acute preclinical toxicity in mice, intraperitoneally. Literature data has reported that if the LD
50 of the test substance is three times more than the minimum effective dose, the substance is considered a good candidate for further studies [
29].
To evaluate the in vivo antitumor and toxicological effects of HE-02, we used the Ehrlich ascitic carcinoma model, an undifferentiated and originally hyperdiploid carcinoma, with high transplantation capacity, non-regressive, rapid proliferation, short duration of life, 100% malignant and non-invasive [
30]. After nine days of treatment with HE-02, it can be observed that there was no change in tumor volume, suggesting that HE-02 was not able to reverse tumor-induced peritoneal ascites. However, significant inhibition of tumor growth was observed considering the cellular viability parameter, especially at 12.5 and 25 mg/kg. As no significant difference was observed between these doses, 12.5 mg/kg dose was selected for the evaluation of possible mechanisms of action of HE-02. Data from the literature show that
Piper alkaloids have significant in vivo antitumor activity against the Sarcoma 180 cell line. In these studies, piperine and piplartine showed antitumor activity at 50 and 100 mg/kg dose [
24].
Regarding cell cycle analysis, HE-02 induced only slight sub-G1 peaks increase (10% higher than the control group), suggesting that, in vivo, the substance does not act by inducing changes in the cell cycle profile. Literature data show that piperine was able to induce G0/G1 cell cycle arrest in prostate cancer cells of the LNCaP, DU145 and PC-3 lines [
31].
Antiangiogenic therapy is a strategy to preventing the growth of new vessels that supply oxygen and nutrients so that tumor cells can proliferate continuously and produce metastasis [
32]. Considering that HE-02 could reduce the microvessel density, it may be suggested that this substance exerts its antitumor activity, at least partially, via antiangiogenic mechanisms. Similarly, data from the literature show that piperine has antiangiogenic effects [
22].
Several mediators contribute to angiogenesis during tumor development, including cytokines and chemokines [
33], reactive oxygen species (ROS) [
34] and NO [
35]. IL-1β is produced by inflammatory cells and, together with TNF-α, INF-γ and IL-18, activate macrophages and neutrophils for phagocytosis and release of reactive oxygen and nitrogen species [
36]. Considering the increase in the concentrations of IL-1β and TNF-α observed after treatment with HE-02, it is possible to infer that this compound modulates the inflammatory response, which may explain the antitumor effect of HE-02 from the activation of macrophages and neutrophils that would be producing ROS and NO to induce cytotoxicity against tumor cells. Piperine also produces immunomodulatory effects. Piperine inhibits transcription factors, such as NF-κB [
37], reduces the expression of INF-γ in human peripheral blood mononuclear cells [
38], and reduces IL-12 in bone marrow-derived dendritic cells stimulated with LPS [
39]. For IL-12, considering its antiangiogenic effect [
40], we can suggest that the decrease on peritumoral microvessel density after HE-02 treatment was induced by IL-12 increase. Furthermore, IL-12 is a cytokine that orchestrates the Th1 type immune response, cytotoxic [
41].
It is known that the anti-inflammatory cytokines IL-10 and IL-4 are involved in the maintenance of Th2 response profile that, with consequent suppression of the Th1 response, produces a microenvironment favorable to tumor growth [
42]. In the present study, HE-02 promoted the reduction of IL-10 and IL-4 levels, suggesting that it promotes the polarization of the Th1 profile.
In addition, HE-02 induced oxidative stress, which suggests that the accumulation of ROS is involved in cytotoxicity against tumor cells. Data from the literature corroborate with these data obtained experimentally for HE-02, since they demonstrate that piperine and analogues induce oxidative stress in tumor cells. For example, piperine, in SK MEL 28 cells, was able to induce DNA damage and cell cycle arrest by inducing ROS production [
21].
Nitric oxide has cytotoxic activity, and is produced by natural killer cells, macrophages and Kupffer cells. In addition to direct cytotoxic activity, NO also promotes suppression of DNA synthesis and regulation of apoptosis [
16]. The data showed that HE-02 promotes its cytotoxic activity also by the stimulation of NO production. These data corroborate previous data on the quantification of cytokines, mainly IL-1β, TNF-α and IL-12, which together suggest, once again, that the cytotoxic activity of HE-02 is probably due to stimulation of the immune system, polarizing a Th1 profile.
The possible toxic effects of HE-02 on the antitumor treatment were also investigated. Considering that HE-02 induced reduction in water and feed intake as well as, a reduction in the final weight of the animals, it may be suggested that this piperine analogue induces a possible gastrointestinal and/or metabolic toxicity, which needs to be better characterized.
Chemotherapy agents that cause hepatotoxicity produce a predictable pattern of injury where the mechanism is direct or idiosyncratic [
43]. However, HE-02 was not able to alter the plasma concentration of the liver enzymes AST and ALT, keeping them within the normal values for the mice of this species, which suggests that HE-02 does not induce hepatic damage in the evaluated conditions.
The biochemical data of AST and ALT are supported by the absence of alteration in liver index, and by the absence of evidence of severe damage to hepatocytes assessed by histological analysis. However, congestion of the branches of the portal vein was observed, which is related to portal pressure. Several factors may alter portal congestion index, ranging from increased portal vein pressure, intrahepatic resistance, increased portal blood flow, and portosystemic collateral circulation. In general, this condition can lead to ascites [
44]. This data suggests that the absence of HE-02 effect on intra-abdominal volume, even with the reduction of viability of tumor cells, may be related to ascites induced by hepatic damage, that is, to this portal congestion. Data from the literature have shown liver toxicity for piplartine and piperine, characterized by Kupffer cell hyperplasia, portal tracts and centrolobular venous congestion, infiltrate of inflammatory cells, microvesicular steatosis, intense ballooning degeneration of hepatocytes and sinusoidal hemorrhage [
24].
Nephrotoxicity is an inherent adverse effect of certain antitumor agents in several ways [
45,
46]. The main clinical utility of urea appears to be in the determination in conjunction with creatinine [
47,
48]. Herein, HE-02 induced only increase in urea plasma concentration. Then, it cannot be confirmed that HE-02 can cause kidney damage. The renal histological study corroborated the biochemical data, and the data of absence of alteration in the kidney index, indicated the preservation of renal structures. Literature data demonstrate that the administration of 100 mg/kg of piperine is able to produce discrete changes in the proximal tubular epithelium, and tubular and proximal hemorrhage [
24].
HE-02 induced spleen hypotrophy in the model tested. This result may demonstrate a decrease in the population of periarteriolar cells of the lymphoid sheath, as occurs with the animals treated with piperine. This may represent a reduced B lymphocyte activity, leading to a reduction of antibody titers [
49]. In addition, treatment with HE-02 reduced thymus mass, corroborating literature data for piperine [
49,
50]. Furthermore, piperine causes a decrease in the cell population of the cortical region of the thymus, leading to a suppression of the maturation of T lymphocytes [
49]. Based on these findings, it can be suggested that HE-02 can induce a suppression of cellular differentiation of the thymus, thus leading to a decrease in thymic mass.
Changes in leukogram level for HE-02 included leukocytosis, accompanied by reduction of lymphocytes and increase of neutrophils. Leukocytosis and consequently neutrocytosis may be related to the cytotoxic mechanism of HE-02. Lymphopenia, in this case, corroborates with thymus mass data, since these data are complementary to the reduction of lymphocyte maturation in this lymphoid organ. In contrast, piperine led to a reduction in leukocyte concentration [
49]. These data demonstrate that piperine analogues may have different hematological toxicity profiles.
In summary, we have obtained a novel piperine analogue which showed antitumor potencial via antiangiogenic and immunomodulatory mechanisms in the sense of inducing a cytotoxic Th1 response. Additionally, we characterized that the replacement of piperidine by an amidoester in HE-02 reduced its toxicity compared to the parent compound. Then, these data support the performance of further preclinical studies with a view to contribute to the discovery of new antitumor drug candidates.