Successful therapy of metastatic melanoma represents one of the main challenges of chemotherapeutic intervention in the field of cancer control. Although clinical protocols including new biological approaches (e.g. targeted agents, immunotherapy) gave some encouraging results, their efficacy and durable responses remain limited and new evidence indicates their use in combination with chemotherapy [
2,
38]. Thus, the search for novel agents capable of exerting anticancer activity appears to be still mandatory. Of great interest, drug repositioning has been growing in importance in the last few years as, by passing much of the early cost and time needed to bring a drug to market, provides a number of low-cost non-cancer drugs for cancer treatment to be exploited in novel anticancer strategies with high therapeutic potential and low-toxicity [
39], allowing also access to cures for a higher population of patients. Among these are antimalarials, a class of compounds that have been proposed as anticancer agents thanks to their anti-proliferative activity since 1953 [
40]. The reappraisal of one these drugs, Pyr, stems from the encouraging results obtained in the treatment of melanoma and other tumors [
7,
8,
10,
11]. In addition, Pyr is already used in humans as an orally administered drug for the treatment of infections caused by protozoan parasites. Of note, Pyr belongs to the group of antifolate drugs that blocks the enzyme dihydrofolate reductase (DHFR). DHFR inhibitors have been studied for many years as anticancer agents for their selective toxicity on rapidly dividing cells such as tumor cells. With this in mind, a series of chemically modified analogues of Pyr has been synthesized and screened in the present work. Here, we report for the first time that one of these, MBP, is a valuable candidate for drug repositioning for cancer treatment as it exerts a powerful effect in both in vitro and in vivo on metastatic melanoma via a mechanism partly overlaying that of Pyr. In particular, multiple effects have been detected: i) apoptosis triggering; ii) activation of cysteine protease, e.g. cathepsin B, activity; iii) inhibition of cell cycle progression, and iv) inhibition of DHFR activity. Of note, MBP activity results associated with the activation of caspase cascade, as either apical caspase (caspase 8–9) or executioner caspase-3 and with the activity of cathepsin B. Interestingly, this lysosomal cysteine protease has been hypothesized for many years as a further actor in the cell death program execution [
41‐
43]. Concerning the cell cycle, the blocking of S-phase and its progression clearly represent an important cytostatic activity of MBP, as for other anticancer drugs [
44]. This property could be of great relevance to develop powerful antitumor combination treatments with drugs able to affect cancer cells in S-phase, such as 5-fluorouracil [
45]. In this regard, the inhibition of DHFR, probably at the basis of the block in S-phase, could be of relevance since folates are key determinants of cell proliferation and represent essential targets for the control of cancer cell growth, as it appears for the prototypical DHFR inhibitor methotrexate, which nevertheless is endowed with high toxicity [
9]. Most importantly, the dose needed for significant in vivo anticancer activity of MBP appears to be about 5-fold lower than that of Pyr, suggesting that lower doses of MBP could show adequate efficacy and cause minor adverse effects compared to the group of antifolate drugs already in use, such as Pyr and, mainly, methotrexate.
A further key point to be considered in anticancer treatments concerns the shift between cell death and cell survival, sustained by apoptosis and autophagy, respectively. Although the role of autophagy in cancer cells is still controversial, recent studies have established that autophagy can be activated to promote the survival of tumor cells when these are exposed to cellular stress conditions such as, radiation, chemotherapy or targeted agents [
46]. Autophagy is thus one of the mechanisms that cancer cells have developed to evade therapy-induced cell death. Apoptosis and autophagy are closely intertwined processes and the shift apoptosis/autophagy is extremely delicate and highly dependent on metabolic interactions [
33]. Previously, we reported that Pyr (as TMZ and other chemotherapeutic drugs) is able to induce autophagy at the early stage of treatment of tumor cells [
33,
47,
48]. In this context, the Food and Drug Administration (FDA) have currently approved the use of CQ in combination with conventional therapies to counteract the mechanisms of cell survival in cancer treatment (source
http://www.fda.gov). Therefore, based primarily on the ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as adjuvant therapy in multiple clinical trials for cancer treatment [
47], leading to their use as potential chemotherapy and radiotherapy sensitizers rather than antineoplastic. In agreement with these findings, when we combined CQ with Pyr, we found an enhanced cytotoxic effect and block of the autophagic flux. In contrast, in our experiments, MBP did not significantly increase autophagy in comparison with baseline levels found in control samples and its combination with CQ did not modify the apoptotic rate of treated cancer cells. Altogether, our data suggest that Pyr could promotes a limited efficacy in vivo, due to its double-edged activity, which induces both apoptotic cell death and autophagy, therefore, limiting its antitumor activity. Conversely, MBP exerts its strong antitumor activity, probably, by stimulating pro-apoptotic effects only [
33]. In addition, as already proposed for antimalarial drug Pyr, the strong efficacy of MBP at very low concentrations suggests that the repositioning of these “low-cost drugs” in cancer chemotherapy could be beneficial.
In a near future, the evaluation of the in vitro and in vivo effects of MBP in combination with new melanoma treatment options appears as mandatory. In particular, further understanding of how MBP could interact with immunotherapy and targeted therapy could present an opportunity for treatment of human metastatic melanoma.