The effects of 1,4-dimethylpyridine in metastatic prostate cancer in mice

Prostate cancer is the second most common cancer of men, affecting approximately 14% of patients [1]. While the risk of developing prostate cancer might be beneficially influenced by proper diet and physical activity [2], there are no confirmed pharmacological means for the prevention of these types of tumors. The majority of prostate cancer cases are diagnosed at the localized stage enabling effective treatment; however, a significant fraction of patients develops metastatic disease that often progresses into treatment-irresponsive, ultimately resulting in patient’s death [3].

Initial treatment of prostate cancer usually comprises hormone therapy; however, when tumors are irresponsive to hormonal treatment (i.e., in case of castrate-resistant prostate cancer), the most common first-line treatment includes the simultaneous use of docetaxel and prednisone. Docetaxel is a semi-synthetic taxane that inhibits microtubular depolymerization and block bcl-2 and bcl-xl gene expression [4, 5]. Prednisone, in turn, is a glucocorticoid that is used to improve symptoms such as pain [6]. It was also shown to inhibit cell proliferation and induce apoptosis in prostate cancer cells [7, 8], and thus decrease the level of prostate-specific antigen [9]. Accordingly, in multiple studies, prednisone was shown to promote anticancer activity of docetaxel [10‐14]. However, the use of glucocorticosteroids in patients with prostate cancer is associated with the risk of adverse side effects (as reviewed, for example, by Dorff and Crowford [15]), and it eventually leads to the development of resistance to chemotherapy [16]. Therefore, there is still an urgent need for new treatment regimens that would enable efficient yet safe means for the therapy of patients suffering from prostate cancer.

1-methylnicotinamide (1-MNA) is an endogenous metabolite of nicotinamide (NA) that has recently gained attention due to its anti-inflammatory [17] and anti-thrombotic [18] activity driven by mechanisms dependent on prostacyclin (PGI₂) release [18, 19]. Another compound that has been shown to modulate thrombus formation based on the PGI₂-related mechanisms is 1,4-dimethylpyridine (1,4-DMP) – a structural analog of 1-MNA that arises naturally in roasted coffee seeds [20]. In addition, it has been recently shown that both 1-MNA and 1,4-DMP could inhibit metastases formation in the model of experimental and spontaneous metastasis of 4T1 murine mammary gland cancer [21].

The present work is aimed to establish whether 1,4-DMP may have an anti-oncogenic effect in the prophylaxis and the treatment of prostate tumors.

1-MNA is an endogenous metabolite of NA that was previously shown to possess significant anti-inflammatory and anti-thrombotic activity [17, 18]. 1-MNA is synthetized by nicotinamide N-methyltransferase (NMMT), an enzyme expressed primarily in liver cells where it participates in methylation of NA and other pyridine compounds [24]. Concurrently, the expression of NMMT was reported in multiple types of cancer in which it was associated with tumor-promoting activity [25‐27] that could be further attributed to 1-MNA [28]. On the contrary, some of the published reports show the beneficial correlation between NMMT expression and cancer survival [29, 30]. Along the lines with such data in a recently published study, we have shown that exogenous 1-MNA does not enhance the growth of cancer cells neither in vitro nor in vivo but, in contrary, may possess antimetastatic activity, most likely resulting from its PGI₂-releasing capacity. We have also shown that 1,4-DMP, a structural analog of 1-MNA, possesses similar antimetastatic activity; however, both compounds seemed to have different mechanisms of action that ultimately resulted in platelet-dependent metastasis inhibition. Importantly, pyridine compounds, but particularly 1,4-DMP, when given in a combination with cyclophosphamide contributed to its anticancer activity enhancing both antitumor and antimetastatic activity of cytostatic drug [21]. Referred studies were, however, carried out exclusively in the mouse model of breast cancer, and the report did not mention any activity of 1-MNA or 1,4-DMP in other types of malignant tumors or with different anticancer agents.

In the present work, we investigated the activity of both compounds in the model of TRAMP mice that spontaneously develop prostate tumors. Similar to our previously reported studies [21], both compounds, when administrated alone to mice developing prostate tumors, revealed no significant anticancer activity; however, to some extent these compounds delayed the disease onset (Fig. 1a–c). Such a delayed disease onset might be attributed to the prostacyclin-dependent activity of the compounds, as prostacyclin was shown to inhibit lung tumor development in PPARγ-dependent mechanism [31] that was also shown to be involved in tumor growth arrest in prostate tumors [32, 33]. Lack of the significant tumor preventing activity of both pyridine compounds while being somehow disappointing in terms of the possible application of 1-MNA, and its analog in the prevention of prostate cancer, is important for their implementation in anticancer treatments, in general, as once again we demonstrated that neither 1-MNA nor 1,4-DMP promoted the growth of solid tumors. On the contrary, we have not observed any antimetastatic activity of neither of the studied compounds. In contrast, among 1-MNA-treated animals, we even observed a slight increase of metastases frequency (Fig. 1c and d). Such a surprising result might be the consequence of prostacyclin-related inhibition of natural killer cells [34] that, in turn, was shown to stimulate prostate tumor metastasis [35]. Another explanation of the observed limited antimetastatic activity of both 1-MNA and 1,4-DMP might be associated with previously reported relationship between thrombin generation and the growth and metastasis of prostate tumors in TRAMP mice [36]. Possibly, thrombin as a potent coagulation and platelet activator that was proven to facilitate metastasis [37] counteracts a possible antiplatelet activity of pyridine compounds in this model. Importantly, in this study, we noted that during the prostate tumor development, TGF-β1 plasma concentration in TRAMP mice increased (Fig. 1p), which remains consistent with the previous reports indicating the usefulness of this molecule as a prognostic factor in prostate tumors [38].

When investigating the anticancer activity of therapeutic regimen including simultaneous use of docetaxel and 1,4-DMP (that seemed to be more potent in the model of prostate tumor as compared with 1-MNA) in the therapy of metastatic human prostate cancer PC-3M-luc2, we observed 60% enhancement of the antitumor activity of docetaxel given alone (Fig. 2c) and complete abolition of metastases formation (Table 3) in mice treated with docetaxel administrated with 1,4-DMP. These results confirm that 1,4-DMP may promote anticancer activity of various cytotoxic drugs. Beneficial therapy outcome was also reflected in the decreased plasma level of TGFβ-1, a molecule often acknowledged as a prognostic marker in prostate cancer (Fig. 2i).

TGF-β1 is commonly recognized as a molecule inducing epithelial-to-mesenchymal transition (EMT) in tumor-forming cells. EMT is a phenomenon in result of which non-invasive tumor cells of epithelial phenotype acquire mesenchymal properties and become able to migrate and invade distant tissues [39]. Therefore, in our study, lower plasma concentration of TGF-β1, and by implication lower metastatic capacity, was associated with higher expression ratio of E-cadherin to N-cadherin (Fig. 2f), cell adhesion molecules commonly accepted as important markers of EMT in cancer cells, including those of prostate origin [40]. Lower metastatic capacity of tumor-forming cells was additionally accompanied by the lower level of short E-cadherin fragments (40 kDa) observed in the tumor mass of mice lacking metastases. Such short intracellular protein fragments arise because of full-length E-cadherin cleavage resulting in the release into extracellular matrix and next to bloodstream of 80 kDa E-cadherin extracellular domain [41]. Indeed, 80 kDa fragments identified in metastatic sites or serum were previously discussed as potential prostate cancer progression markers [42, 43]. Accordingly, in our study, we observed that 40 kDa intracellular domain of E-cadherin was abundant in tumor mass isolated from mice bearing PC-3M-luc2 tumors diagnosed with metastases (Fig. 2g). Finally, the prominent efficacy of the combined treatment comprising the use of docetaxel and 1,4-DMP is additionally confirmed by the decreased expression of VEGFR-1 (Fig. 2h), another prognostic marker that has been previously linked to enhanced metastatic potential of prostate tumors [44].

We have previously shown that observed enhanced antitumor and antimetastatic activity of cytotoxic drugs when in a combination with 1,4-DMP might be a result of anti-platelet activity of the latter compound [21]. Platelets, in turn, contribute to metastases formation by several mechanisms as comprehensively reviewed in the literature [45, 46]. To confirm that increased anticancer efficacy of docetaxel observed when cytotoxic drug was given with 1,4-DMP was associated with diminished platelets activity, we have analyzed morphological and biochemical parameters reflecting platelet activation status. In this regard, in mice receiving the studied combined treatment, we observed lowered values of mean platelet volume and PDW (Fig. 3a–d) that may suggest decreased platelet activity [47, 48]. Additionally, in mice treated with docetaxel and 1,4-DMP, we also noted a marked reduction in plasma concentrations of TXB₂, vWF and soluble P-selectin (Fig. 3e–h), constituting biochemical markers, further confirming diminished platelet activity.

Interestingly, increased antitumor activity of docetaxel when administrated simultaneously with 1,4-DMP was accompanied by its reduced toxicity manifested in the decreased incidence of treatment-related deaths and improved liver function (Fig. 4). Although we are currently investigating these phenomena, our initial results indicate that the observed protective activity of 1,4-DMP may involve acetylcholinesterase and consequently histamine-dependent pathways. It seems possible that in response to the treatment with 1,4-DMP, the level of histamine is increased that, in turn, may prevent liver injury [49]. This novel and unexpected feature of the 1,4-DMP treatment might not only be of a great value for possible improvement of side effects in patients undergoing chemotherapy, but may also allow to increase dosages in patients with drug-resistant tumors to induce desired response while maintaining acceptable treatment toxicity.

The results of the presented study prove that neither 1-MNA nor 1,4-DMP when administrated alone do not influence the development and growth of the primary prostate tumors supporting our previous findings in the murine model of metastatic breast cancer. However, pyridine compounds, such as 1,4-DMP, may beneficially influence the antitumor and antimetastatic activity of docetaxel and additionally limit the side effects accompanying chemotherapy. Such findings allow us to believe that pyridine compound endowed with PGI₂ releasing properties [18, 20] may become a promising agent for the adjuvant therapy of metastatic cancer.