**4. Discussions**

The endocannabinoid system is currently under intense investigation due to the therapeutic potential of endocannabinoids as treatment options for cancer. Structural modifications of these substances are under investigation and synthesis of novel derivatives with better properties is being explored. Therefore, the aim of this study was to investigate

the anti-tumorigenic effect of HU-585, a novel compound obtained by a combination of features of the endocannabinoid anandamide and the drug Minerval (HU-600), expecting to potentiate the antitumorigenic effects of HU-600 against neuroblastoma, an aggressive and resistant pediatric tumor in which identification of new therapeutic strategies are needed.

Minerval, a nontoxic synthetic analog of oleic acid (OA), represents a new class of orally bioavailable lipids used for membrane lipid therapy (MLT). MLT is a new rapidly evolving approach for treating cancer, in which the cellular membranes rather than specific proteins constitute the therapeutic target [22].

In the search for molecules capable of regulating membrane lipid structure, oleic acid was found to be the most active in many types of cancers [24]. However, the therapeutic effect of oleic acid is limited due to its rapid metabolism [25]. In contrast, its synthetic analog, Minerval, is believed to have a more long-lasting pharmacological effect, which favors its therapeutic effect [26]. Minerval has been shown to restore the normal membrane lipid structure and composition in certain tumor cells [27] and by this to inhibit membrane protein-associated aberrant signaling pathways, such as RAS/MAPK and PI3K/AKT pathways [28]. By contrast, Minerval does not significantly alter membrane lipid composition in non-tumor cells, which explains its specificity for cancer cells and the lack of undesired side effects [29]. Moreover, the difference in IC50 values between normal cells (>5000 μM) and cancer cells (30–200 μM) and minimum lethal dose >3000 mg/kg in rats indicate that the therapeutic window for this drug is far below the maximum tolerated dose (or minimum lethal dose), unlike most anticancer drugs currently used. These facts support the specificity of Minerval and its use as a therapy agen<sup>t</sup> to treat cancer. This efficacy and lack of toxicity at therapeutic doses has been acknowledged by the European Medicines Agency (EMA) to designate 2OHOA orphan drug for the treatment of glioma [16].

In a similar way, endocannabinoids, such as AEA are lipid-based derivatives that demonstrate anti-tumorigenic effects mediated by modulation of the ERK and AKT signaling pathways [30,31]. We have previously shown that the ethanolamides of fatty acids have a better anticancer profile than the acids themselves [32]; hence, we synthesized and tested the ethanolamide derivative of Minerval. The novel compound, chemically closely related to Minerval, also resembles the endocannabinoid anandamide (AEA) found in the mammalian body [33]. As Minerval and AEA exert similar protective effects against cancer, we assumed that a molecule that is a derivative of both may have a better anticancer effect while maintaining the high safety profile typical of these two compounds.

To investigate the anti-tumorigenic effects and the mechanisms behind the effects of Minerval (HU-600) and the novel derivate HU-585 on SK-N-SH NBL cell line, cell viability, CFU, and migration in response to treatment were first analyzed followed by apoptosis and senescence studies.

We found that treatment with either HU-600 and HU-585 had an antiproliferative effect as demonstrated by MTT and by CFU assays. HU-600 had only a modest effect on the viability of these cells with a significantly better effect of the derivate HU-585. In addition, as membrane lipid composition has been shown to influence cancer cell migration abilities [34–36] we next explored whether HU-600 and its derivative HU-585 can disrupt the migratory ability of NBL cells, a crucial step in the metastatic process and in tumor dissemination. Using migration assay, we found that HU-585 had a better antimigratory effect on NBL cells in comparison to HU-600. Therefore, the conversion of HU-600 to its ethanolamide, leading to HU-585, increases the pharmacological potency of this drug, regarding viability and migration ability of NBL cells.

Impaired apoptosis plays an important role in tumorigenesis and tumor resistance to oncologic treatment [37]. The mechanism of apoptosis is evolutionarily conserved and is executed by a family of proteins called caspases, whose activation is mainly regulated by the anti- apoptotic Bcl-2 family proteins, including Bcl-2, Bcl-XL, Bcl-w, Mcl-,1 and Bfl-1/A1 [38]. HU-600 has been shown to induce apoptosis in several cancer cell lines [39], hence we sought to explore apoptosis as a possible anti-tumorigenic mechanism of its derivative

HU-585. We showed that HU-585 induces apoptosis as demonstrated by increased levels of active caspase-3 and a decreased expression of Bcl-2, both markers of apoptosis [40–42].

Bcl-2 expression was reported to be strongly increased in most NBL tumors, supporting that Bcl-2 antagonists may have clinical utility for a large subset of patients [43]. Indeed, preclinical studies using Bcl-2-specific inhibitors have demonstrated anti-tumor activity in neuroblastoma tumors with high Bcl-2 levels. Currently, Bcl-2 inhibitors in combination with classical cytostatic drugs are being investigated in clinical trials for the treatment of relapsed or refractory neuroblastoma [44].

Many oncogenic stimuli leading to apoptosis can also induce senescence, which is a special state of durable cell cycle arrest. Hence, further to our observation related to the proapoptotic properties of HU-585, we decided to explore whether HU-600 and HU-585 also induce senescence in SK-N-SH NBL cell line. Our results demonstrate that cells treated with HU-585 exhibited senescence in contrast to cells treated with HU-600. Senescence is generally regarded as a tumor suppressive process which evolved alongside apoptosis to suppress tumorigenesis and tumor progression and is considered as an important alternative cell fate to apoptosis [45,46]. Thus, one way to enhance anti-cancer treatment is to use compounds that induce senescence. To this end, senescence-inducing compounds have been developed, including CDK4/6 inhibitors, such as abemaciclib, palbociclib, and ribociclib [47,48]. With respect to neuroblastoma, LEE011, a highly specific CDK4/6 inhibitor caused cell-cycle arrest and cellular senescence in a large subset of neuroblastoma cell line and Xn models [49]. Because this class of drugs has shown promise in treating several cancers in pre-clinical and clinical studies [47–49], high-throughput screens have been employed to find additional drug targets that trigger senescence in cancer cells [50]. Based on our present findings, we think that HU-585 can also be considered as a senescenceinducing compound and is a promising candidate for further testing and implementation in current treatment protocols of neuroblastoma patients.

Paradoxically, senescence has also been proposed to have pro-tumorigenic effects. Accumulating evidence indicates that, following treatment, senescent tumor cells promote tumor relapse, aggressiveness, and metastases via upregulation of antiapoptotic mechanisms [51] and by secretion of cytokines and growth factors that may promote the proliferation of tumor cells [52]. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, known as the senescence-associated secretory phenotype (SASP), which may have proneoplastic properties, including angiogenesis, epithelial—mesenchymal transitions, and differentiation within the local microenvironment [53–56].

Although senescence induction in cancer cells is a potential therapeutic option to reduce initial tumor growth, it seems to be an imperfect tumor-suppressive treatment. Hence, we assume that chronically persisting senescent cells should be removed by senolytic drugs, which selectively destroy such cells. These drugs can presumably be given in combination with other cancer therapies in order to minimize progression risk and avoid deleterious side effects [57].

It has been shown that overexpression of Bcl-2 counteracts the pro-apoptotic genes during senescence [58]. Senolytic drugs, which target the anti-apoptotic signaling through Bcl-2 family members, (Navitoclax/ABT-263 and ABT-737), were shown to be effective in inducing cell death in senescent cells [59]. As manipulation of the anti-apoptotic Bcl-2 family proteins can influence the choice between senescence and apoptosis, we decided to explore whether treatment with the combination of HU-585 with anti Bcl-2 compounds can have a better anti-tumorigenic effect compared with each drug alone in vitro and in vivo. We have shown that treatment of SK-N-SH NBL cells with a combination of anti Bcl-2 compounds and HU-585 resulted in enhanced anti-proliferative effect in vitro and reduced Xns growth in vivo in comparison to treatment with HU-585 alone.
