Compounds that act as agonist for TLRs, or in other words, compounds or molecules that bind to and activate TLRs are the subject of intensive research and development for the treatment of infectious diseases and cancer. In this regard, the present studies detail a series of in vitro and animal models and demonstrate that the immunomodulator P-MAPA exhibits significant antimycobacterial and antitumor activity. TLR activation facilitates and instructs the development of adaptive immune responses by increasing the levels of expression of co-stimulatory molecules such as CD80 and CD86 on DC, allowing DC to more effectively activate T cells [21
An important aspect of the induced cytokine production is the differentiation of T cells either into Th1
subsets, which will guide the pattern of adaptive response launched by the host against the pathogen. For instance, human monocyte-derived DC stimulated with lipopeptide from Mycobacterium tuberculosis
secretes IL-12 over IL-10, skewing the host's adaptive immune response toward a Th1
pattern, characterized by a cellular, cytotoxic T cell response [33
In TB, recent evidence shows that the TLRs are able to recognize Mycobacterium tuberculosis
-associated molecular patterns and mediate the secretion of cytokines and other antibacterial effector molecules [34
]. The immune system recognizes pathogen-associated mole cular patterns (PAMPs) such as the bacteria’s outer cell wall composition of peptidoglycan, mycolic acids, lipomannan, and lipoarabinomannan [36
]. PAMPs stimulate immune recognition receptors such as toll-like receptors (TLR) [37
]. In vitro studies have delineated these mechanisms, providing evidence that TLR2 and TLR4 are stimulated by interactions with Mycobacterium
cell wall molecules leading to the maturation of DC, macrophages, and production of IFN-γ [39
]. Other studies suggest that TLRs variants may contribute to human susceptibility to tuberculosis disease [41
]. Recent studies in animal models also suggested that TLR, mainly TLR-2 and TLR-4 may play a protective role in host defense against lung infection by Mycobacterium tuberculosis
The protective immunity against Mycobacterium tuberculosis
has been ascribed to CD4+ T cell-mediated immunity [42
]. The breakdown of immune responses designed to contain the infection can result in reactivation and replication of the bacilli, with necrosis and damage to lung tissue [44
]. An efficient immune response against the intracellular pathogen Mycobacterium tuberculosis
is critically dependent on rapid detection of the invading pathogen by the innate immune system and the coordinated activation of the adaptive immune response [45
]. An effective response against Mycobacterium tuberculosis
was demonstrated in vivo in the present study. P-MAPA did not show any direct in vitro antimicrobial activity. In vivo P-MAPA alone or combined with MXF did not show synergistic and/or antagonic effects; however P-MAPA induced a significant antimicrobial response against Mycobacterium tuberculosis.
Future studies are required to investigate whether this result is observed with the use of P-MAPA combined with other TB drugs (such as rifampin or isoniazid), or whether this is a MXF-specific effect. This finding could be relevant for the use of P-MAPA as candidate to be evaluated in antitubercular treatments, with the aim of reducing the doses of antimicrobials or shortening the period required for administration of such drugs.
Ayari et al. [46
] have recently shown that TLRs are expressed in normal urothelium and BC. The role of TLRs in cancer is a matter of debate because conflicting data argue that TLRs are negative or positive regulators of cancer [47
]. Different authors demonstrated that TLRs help tumour cells evade immune system response [12
] while others showed that TLRs expression on tumour cells might drive them to apoptosis or other types of cell death [50
]. Activated TLRs on cancer cells may promote cancer progression, invasion, anti-apoptotic activity and resistance to host immune responses [49
]. Some TLRs have been connected with tumor progression and invasion as after TLR stimulation they can increase the expression of matrix metalloproteinases, up regulate different cytokines and chemokines or other inflammatory factors [54
]. Matijevic et al.
] and Matijevic & Pavelic [58
] investigated the difference between primary tumour and metastasis based on TLR3 level and found a huge difference between FaDu and Detroit 562 cell lines, which were of the same origin (pharynx), but FaDu was primary and Detroit 562 metastatic carcinoma. The same authors reported on the dual role of TLR3 in pharynx metastatic cell line (Detroit 562); on the one hand TLR3 activation drove cells to apoptosis, while on the other its stimulation contributed to tumor progression by altering the expression of tumor promoting genes (PLAURRORB
) and enhancing the cell migration potential. In addition, these authors showed that TLR3 signaling pathway was functional in another metastatic cancer cell line (SW620) suggesting TLR3 might be important in the process of tumor metastasis.
In this context, the administration of TLR agonists was also reported to exert strong antineoplastic effects against established tumors in mice and humans [12
]. TLR activation may also cause tumor regression by increasing vascular permeability and through the recruitment of leukocytes, which determines tumor cell lysis by natural killer
(NK) and cytotoxic T cells [59
]. Accordingly, one of the most promising effects of TLR stimulation by specific agonists in cancer therapy is the activation of the adaptive immune system [14
The contradictory evidence that TLR promotes carcinogenesis, whereas in others it exerts antitumor effects, could be explained by the different intensity and nature of the inflammatory response [48
]. In fact, chronic inflammatory processes are milder than acute inflammatory responses, which are aimed at inducing pathogen clearance. In most cases, cancer-associated inflammation is similar to chronic inflammation, including the production of factors that stimulate tissue repair and cancer cell survival and proliferation [48
]. However, if the inflammatory response develops into acute inflammation, an immune effector mechanism is activated, and cancer regression takes place [48
]. Among the different elements that control neoplastic processes, a major role is attributed to members of the chemokine superfamily. Chemokines expressed by tumor cells and by host cells play a critical role in determining the fate of the developing tumor by regulating the migration of different leukocyte subtypes [48
]. The relative proportion of each defense cell type (macrophages, T cells, NK cells, dendritic cells, or other leukocyte subtypes) within the tumor largely dictates the immune profile at the tumor site; local production of numerous inflammatory mediators is crucial for the recruitment and activation of leukocytes in addition to macrophages and mast cells [48
]. In particular, CD8 T cells and some types of innate immune cells, such as NK cells, can protect against experimental tumor growth [48
Thus, the present study showed that P-MAPA increased significantly TLR2, TLR4 and p53 protein levels. In addition, it was demonstrated that this immunomodulator was more effective in the treatment of BC compared to BCG. These results were correlated with the ability of P-MAPA to act as TLR ligand, mainly for TLR2 and TLR4. The increased TRL2 and TLR4 levels were fundamental for antitumor immunotherapy of BC.
Furthermore, the present study demonstrated that the MNU animal model could lead to BC, besides kidney failure and increased alkaline phosphatase activity. Proctor et al.
] demonstrated that increased alkaline phosphatase activity was associated with the presence of cancer and could be associated with mortality.
This MNU-Citrate animal model has particular advantages for the experimental analysis of complete carcinogenesis, since the carcinogen can be administered directly in quantifiable pulse doses, via intravesical instillation [66
]. In addition, this autochthonous BC model includes low cost, reproducibility and an immunocompetent host, which is important, for example, when studying intravesical BCG treatment of BC [26
]. Bladders treated with intravesical MNU-Citrate develop progressive neoplastic changes and tumours become progressively less differentiated with time. These lesions progress from hyperplasia, atypia, carcinoma in situ (CIS) and papillary carcinoma to large bulky muscle invasive tumours that completely fill the bladder lumen, obstruct the ureters and kill the animal [26
]. Furthermore, the MNU-Citrate animal model is similar to human urothelial carcinogenesis; it involves the effect of environmental agents (carcinogen, smoking) in a genetically susceptible substrate (Fisher 344 rats) [26
]. Intravesical instillation of fractionated doses of carcinogen and promoter provided a more controlled cancer model than those using carcinogens in the diet or drinking water. Added to its effectiveness (100
% of induction), low cost (not dependent on high-cost technologies) and short period of induction, it is in a position of superiority considering other available models, being a useful model for further studies [26
The ability of intravesically administered BCG to exert an antineoplastic effect on BC is widely accepted and serves as the basis for its clinical use [70
]. This agent induces a complex systemic immune response comprising humoral and cellular components [72
]. Recent studies have demonstrated that polymorph nuclear neutrophils (PMN) migrating to the bladder after BCG instillation release large amounts of TNF-related apoptosis-inducing ligand (TRAIL), along with cytokines that recruit other immune cells, suggesting that PMN play a key role in the antitumor response to BCG therapy [15
]. BCG intravesical immunotherapy induces a well-described T-lymphocyte predominant inflammatory infiltrate in the bladder wall and induces cytokines in the bladder and in urine [74
]. These cytokines have anti-tumor and anti-angiogenic activity, and they share common regulatory pathways [74
]. A number of prior reports documented a direct antiproliferative effect of BCG on urothelial carcinoma cells [75
]. Potential mechanisms contributing to this biological effect include cell cycle arrest and/or apoptosis. DiPaola & Lattime [71
] demonstrated that BCG induced signaling alters the susceptibility of the cell to an apoptotic insult. Thus, the consensus is that BCG serves as an immune potentiator of lymphocytes, namely an adjuvant, via the maturation of DC [22
]. It appears that the effects in bladder cancer are local only. There is no protection against the development of tumors in areas where there is no BCG contact (e.g., the distal ureter and prostatic urethra).
BCG use is limited in BC by treatment failure, adverse effects and intolerance that occur in over two-thirds of all patients and consist largely of irritative voiding symptoms including haematuria, dysuria and urgency [19
]. Even major complications and death related to treatment have been described [76
]. In the BC studies presented here, the results demonstrated that P-MAPA immunotherapy was more effective in restoring normal morphological features and alkaline phosphatase activity compared to BCG. Concerning the toxicological analysis, the present results showed that both P-MAPA and BCG treatments did not show hepatotoxicity and nephrotoxicity compared to MNU group, which showed higher creatinine and urea serum levels; and indicating that high molecular weight of P-MAPA and BCG probably hinder local diffusion into the bladder wall thereby preventing systemic toxicity. Although BCG did not cause systemic toxicity, this immunotherapy led to intense local adverse effects such as haematuria compared to P-MAPA treatment, which was absent.
In addition, P-MAPA also increased the p53 protein level in the BC cancer model. The p53 gene and protein expression levels both play a critical role in the regulation of the normal cell cycle, cell cycle arrest, and apoptotic response [77
]. Alterations in the p53 protein, leading to a loss of its tumor suppressor function, have been reported previously by some authors [79
]. The p53 gene status has been examined in a number of malignancies, including cancers of bladder, breast, lung, ovary and colorectal cancer [79
]. Studies of promoter response element sequences targeted by the p53 master regulatory transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human TLR gene expression [84
]. Most of the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites [84
]. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors [84
]. Also, Menendez et al.
] demonstrated that all TLR genes showed responses to DNA damage, and most were p53-mediated.
In conclusion, the presented results showed that P-MAPA was able to improve, and/or re-establish the immunocompetence when the immune system was impaired, by cancer and possibly in infectious diseases, resulting in remarkable therapeutic effects. The P-MAPA therapy showed stimulatory effect on TLRs and p53 that correlated with the decrease of cancer state. Furthermore, P-MAPA induced significant responses in vivo against TB. Thus, these results pointed out that P-MAPA hypothetically acted in a common control mechanism for both infectious diseases and cancer, which involved TLRs and p53 signaling pathway. Finally, the low toxicity of P-MAPA combined with its significant antimycobacterial and antitumor activities warrant its development as a potential candidate for adjuvant treatment of cancer and infectious diseases.
This work shows the toll-like stimulating properties of P-MAPA in vitro and in vivo. The immunomodulator P-MAPA alone showed significant efficacy against Mycobacterium tuberculosis (Erdman strain)
in vivo when administered at 5
mg/kg. P-MAPA did not show any direct antibactericidal activity in vitro against Mycobacterium tuberculosis
(H37Rv).Taken together data obtained suggest an immunotherapeutic effect of the compound against tuberculosis in vivo. Stimulation on TLRs is a feasible possibility to explain this effect, to be explored in additional studies.
Since the P-MAPA has not shown antagonic effects on the antimicrobial action of MFX in vivo, we aim to assay the compound together with other antitubercular compounds at lower doses of antimicrobials, or by shortening the period of time administration of such medicines. The low in vivo toxicity of P-MAPA is another benefit of the compound. Experiments on P-MAPA activity on Cytochrome P-450 are under way which may provide additional data on the potential for interaction with other compounds.
In a series of experiments evaluating the potential of P-MAPA in cancer models, the toll-like ligand properties of P-MAPA were tested in vitro and subsequently the use of the compound alone and compared with BCG in an animal model when the immune system is impaired by carcinogen and with BC.
The results indicate the stimulatory effect of P-MAPA and BCG on TLR2, TLR4 and p53 in vivo. More importantly, the results correlated with a re-establishment of immunocompetence and a significant therapeutic effect in the treated animals was seen for both compounds in relation to controls. Thus, looking ahead, the data provides instigating insights for a possible use of P-MAPA as adjuvant with BCG or other therapies aiming to boost its effects against cancer.
An important question to be answered concerns the determination of the optimal dose for the immunomodulator P-MAPA to be used alone or in combinations with other drugs. Like other biological therapies, the in vivo response to P-MAPA may be not linear and hence lower doses or single dose schedules may achieve improved responses in vivo. Previous results from experiments concerning the use of P-MAPA in the Punta toro virus (PTV) infected mice model suggests that single dose schedules may work better than repeated doses [25
]. Further studies are being planned to elucidate this question using animal models for the study of infectious diseases and cancer.
The modification of antioxidant enzyme activity that plays an essential role in cellular defense mechanisms against oxygen toxicity has been observed in various cancers. The importance of antioxidants and reactive oxygen species (ROS) in carcinogenesis is considered in experimental studies in vivo. The Nuclear Factor-κB (NF-κB) signaling pathway plays an important role in inflammation, cancer and stress responses. Some NF-κB targets, such as the Cytochrome p450, CYP1B1, antioxidants and, mainly TLRs, have been implicated in modulating cellular redox potential. Thus, studies concerning the TLR and its signaling pathways associated with antioxidants and ROS are being planned to investigate the role of P-MAPA as an antioxidant agent in vitro and in vivo. These studies may provide useful data to complement the understanding of the P-MAPA´s mechanism of action.