Effects of P-MAPA Immunomodulator on Toll-Like Receptors and p53: Potential Therapeutic Strategies for Infectious Diseases and Cancer

Immunotherapy represents an approach for the treatment of infectious diseases and cancer [1–3]. Effective immunotherapy for chronic infectious diseases or cancer requires the use of appropriate target antigens, the optimization of the interaction between the antigenic peptide, the antigen-presenting cell (APC) and the T cell and the simultaneous blockade of negative regulatory mechanisms that impede immunotherapeutic effects [1, 3]. In cancer, besides the impairment of the immunological status associated with the main disease, several therapies can often cause additional immunosuppression creating the conditions for the emergence of infections [1–4]. In this scenario, compounds which can act in the immune system such as new vaccines, vaccine adjuvants and biological response modifiers are considered potential candidates for the treatment of these diseases or conditions. Compounds that target the TLRs may represent starting points for the development of new drugs, since the TLRs are increasingly implicated in the pathogenesis of some diseases [5, 6]. In fact, recent advances in TLR-related research have also shown the therapeutic properties of these receptors against several diseases, including infectious diseases and cancer [7–9]. Consequently, compounds that mimic pathogen associated molecular patterns and activate immune cells via TLRs are candidate drugs being developed to treat several diseases and to be used as vaccine adjuvants. TLRs are transmembrane proteins that recognize pathogen-associated molecular patterns as well as endogenous damage-associated molecular patterns and elicit pathogen-induced and noninfectious inflammatory responses [10, 11].TLRs were initially detected only on immune cells, but recent studies demonstrate that tumor cells express functional TLRs and that TLR signaling can promote opposite outcomes: tumor growth and immune evasion or apoptosis and cell cycle arrest [12–14].

Compounds from microbial sources as well as bacterial strains themselves, such as Bacillus Calmette-Guerin (BCG), are used as therapeutic tools in the treatment of some types of cancer, including urothelial cancer [15, 16]. BCG is a live, attenuated strain of Mycobacterium bovis used widely for tuberculosis (TB) prophylaxis. However, the exact mechanism by which BCG exerts its antitumor effect remains unknown. The treatment of choice for BC is transurethral resection (TUR) and adjuvant therapy with BCG [15–17]. The objective of intravesical BCG therapy is to reduce the risk of recurrence or eradicate carcinoma in situ in patients if complete resection it is not possible [18, 19]. BCG administered as a control in the cancer model admittedly acted as TLR2 and TLR4 agonist, since the cell wall of mycobacteria contains certain antigens which are recognized by local macrophages and immature dendritic cells (DC) [20]. Peptidoglycan is an important mycobacterial cell wall component, which is covalently linked to arabinogalactan and mycolic acids [21, 22]. In a series of experiments, different research studies have demonstrated that this mycobacterial cell wall component stimulated TLR2 and TLR4 responses in immature DC cells [21, 22]. Although intravesical BCG immunotherapy treatment can reduce the risk of recurrence and progression of BC, its use is limited by the adverse effect profile and intolerance that occurs in 20 % of patients, from mild and self-limiting side effects to potentially life-threatening complications, such as systemic BCG infection [23, 24].

With respect to infectious diseases, tuberculosis is far from being controlled or eradicated. On the contrary, it is becoming resistant to existing drugs, including most antimicrobial drugs. Mycobacterium tuberculosis, the bacterium that causes pulmonary TB remains a major global health problem, infecting nearly one-third of the world’s population and killing at least 3 million people every year [4]. The occurrence of drug-resistant strains of Mycobacterium tuberculosi s emphasizes the urgent need for developing new drugs, including those drugs capable of boosting the body’s immune response. Immunotherapy through the use of compounds that act as agonists for TLRs, can represent a valuable approach to infectious diseases and cancer, when used in combination with existing therapies.

P-MAPA can be an immunomodulatory drug candidate. The compound’s ability to fight cancer and infectious diseases was detected in earlier studies using animal models [25–27]. P-MAPA is an acronym for Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride, a proteinaceous aggregate of ammonium and magnesium phospholinoleate-palmitoleate anhydride, with immunomodulatory properties produced by fermentation of Aspergillus oryzae, under development by Farmabrasilis, a non-profit research network [25, 28, 29]. The compound is a nonlinear biopolymer with molecular mass of 320 kDa. The main components of P-MAPA are Mg²⁺, NH⁴⁺, phosphate, linoleic acid, palmitoleic acid and protein [25, 29–31]. The P-MAPA immunomodulator was originally intended for cancer treatment, and has effectively demonstrated antitumor activity in several animal models including cancer by chemical inducers/promoters [27]. P-MAPA induces immunodulatory effects, including increased cytokine production, mainly interferon-gamma (IFN-γ) and interleukin 2 (IL-2), and stimulates nitric oxide release by macrophages [25, 29–31]. These findings expanded the potential therapeutic applications of the compound, suggesting that P-MAPA, like other compounds with immunomodulatory properties, can fight a wide range of infections caused by intracellular pathogens [25, 31]. The compound did not show significant toxicity in the preclinical phase when assessed in in vitro cell toxicity assays (V-79 Chinese hamster cell line at concentration of 120 μg/ml) and in vivo acute and chronic toxicity models using Swiss mice (single dose toxicity, oral, at 30 g/kg,), Wistar rats (12-week multiple-dose toxicity, injected subcutaneously, at 1, 10 and 100 mg/kg), and monkeys (Cebus apella; 4-week multiple dose toxicity, injected intramuscularly at 5,10 and 30 mg/kg) [28, 31]. Moreover, the use of P-MAPA in clinical trial phase I did not show any signs of adverse drug reaction at dosages of 5 mg/square meter, injected intramuscularly, 3 times a week, for 6 weeks [29].

Thus, the present study was aimed to characterize the effects of P-MAPA on TLRs in vitro and in vivo, as well as to assess its potential as adjuvant therapy for infectious diseases and cancer. Regarding its action against cancer, the efficacy of P-MAPA was compared versus BCG in the BC mouse model. And regarding its action against infectious diseases, the in vivo efficacy of P-MAPA was compared alone and co-administered to moxifloxacin (MXF) in a tuberculosis mouse model (Erdman strain).

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, 32].

An important aspect of the induced cytokine production is the differentiation of T cells either into Th₁ or Th₂ 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, 35]. 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, 38]. 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, 40]. 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[42, 43].

The protective immunity against Mycobacterium tuberculosis has been ascribed to CD4+ T cell-mediated immunity [42, 43]. 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, 45]. 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, 48]. Different authors demonstrated that TLRs help tumour cells evade immune system response [12, 49] while others showed that TLRs expression on tumour cells might drive them to apoptosis or other types of cell death [50–52]. Activated TLRs on cancer cells may promote cancer progression, invasion, anti-apoptotic activity and resistance to host immune responses [49, 53, 54]. 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 [54–56]. Matijevic et al.[57] 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 (PLAUR RORB) 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, 14, 59]. 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, 60].

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, 61]. 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, 62]. 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, 63]. In particular, CD8 T cells and some types of innate immune cells, such as NK cells, can protect against experimental tumor growth [48, 64].

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.[65] 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, 67]. 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, 67, 68]. 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, 67, 69]. 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, 71]. 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, 73]. 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 [77–79]. Alterations in the p53 protein, leading to a loss of its tumor suppressor function, have been reported previously by some authors [79, 80]. The p53 gene status has been examined in a number of malignancies, including cancers of bladder, breast, lung, ovary and colorectal cancer [79–83]. 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.[84] 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.