1. Introduction
Cancer still remains a leading public health issue and is a major cause of premature death worldwide, resulting in multiple severe social and economic problems. Moreover, the global tumor burden is predicted to increase within next decades [
1,
2].
Neuroblastoma (NB) is one of the most frequent solid cancers in infants and young children, representing approximately 8–10% of all childhood malignancies [
3,
4]. NB, as an embryonal tumor originated from stem cells of the neural crest, may arise within the sympathetic nervous system in the neck, chest, abdomen or pelvis. Therefore, NB remains a complex disease with diverse clinical and histopathological manifestations [
4,
5]. Recent advanced high-throughput study and large-scale profiling showed several genome, epigenome and transcriptome abnormalities determining high biological and clinical heterogeneity and variability of pediatric neuroblastomas [
5]. Prognoses for patients with NB are quite variable—from spontaneous regression mainly in infants ≤18 months, to aggressive disease, characterized by distant metastases and resistance to standard treatment modalities in older children [
3]. Current treatment options for children with NB are local-control surgical resection, radiotherapy, an immunotherapeutic approach using monoclonal antibodies [
5] and multidrug chemotherapy with irinotecan, topotecan, vincristine-irinotecan [
6,
7,
8] and cisplatin (cis-diaminedichloroplatinum[II], CDDP) [
9,
10,
11]. However, no significant progress has been achieved in terms of survival rates for patients with advanced or metastatic NB.
Pediatric soft-tissue sarcomas represent a very heterogeneous group of malignant tumors of mesenchymal stem or progenitor cell origin, accounting for 7% of all childhood cancers (between 2 to 6 and 10 to 18 years old) [
12]. Among them, the most common type (nearly 40%) is rhabdomyosarcoma (RMS), generally localized in the head, neck and genitourinary system (embryonal RMS, mostly in younger children) or trunk regions (alveolar RMS, in older children) [
13]. The significant histological and biological complexity of RMS was evidenced by whole-genome and transcriptome sequencing of rhabdomyosarcoma cells that revealed frequent genetic aberrations such as chromosomal translocations, allelic loss, gene mutations and fusions. Although the majority of children with localized disease may benefit from multi-modal therapy that improves 5-year survival rates by up to approximately 70%, the outcomes of patients with high-risk metastatic or recurrent RMS are still unsatisfactory [
14]. Currently available standards of treatment include surgery, combination chemotherapy (VAC, vincristine–actinomycin D-cyclophosphamide and IVA, ifosfamide-vincristine-actinomycin D in the USA and Europe, respectively) and/or radiation. However, therapeutic protocol has not changed prominently since the 1980s [
13].
To conclude, there is still a strong need for novel alternative types of chemotherapeutic agents which will overcome cancer cells’ resistance, augment treatment effectiveness and reduce heavy adverse reactions to improve the outcome of pediatric cancer patients.
Betulin (Bet, 3-lup-20(29)-ene-3
β, 28-diol) is a pentacyclic lupane-type triterpenoid which naturally occurs abundantly in the outer bark of birch trees (
Betula,
Betulaceae family) [
15]. Bet has been found to demonstrate a broad spectrum of biological and pharmacological activities, among which, its chemopreventive and anti-tumor activities attract the most attention [
16,
17]. Several in vitro studies have evidenced the significant inhibition of cancer cells’ viability/survival, growth and proliferation [
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32], migration [
18], angiogenesis [
24,
31,
33,
34], as well as perturbation in cell cycle progression [
31,
35] and the induction of apoptosis [
18,
20,
23,
24,
29,
31,
36,
37] after treatment with Bet. What is more, its promising activity has also been evidenced in animal models [
24,
31,
34,
38,
39]. As a result of its multiple anti-cancer activities, selective cytotoxicity towards cancer cells and relatively low toxicity against normal cells [
18,
25,
40,
41,
42,
43], Bet has also been utilized as a precursor compound for the synthesis of numerous novel derivatives. The structure of the Bet molecule enables several chemical modifications and consequently, many of its derivatives with improved biological and pharmacological properties can be synthesized for potential implementation into clinical oncology [
17,
44,
45].
A series of Bet derivatives bearing an alkyne moiety at carbon C-3 and/or C-28 (acetylenic synthetic Bet derivatives, ASBDs) were synthesized, and the procedure as well as structural analysis and comprehensive chemical characterization were published [
25]. In this study, we aimed to investigate the anti-tumor potential of two ASBDs, 28-
O-propynoylbetulin (EB5) and 28-
O-propargyloxycarbonylbetulin (EB25/1), in both NB and RMS pediatric cancers in vitro. Additionally, we also calculated physicochemical parameters of the analyzed ASBDs and predicted their druglikeness, medicinal chemistry friendliness and ADMET profile (absorption, distribution, metabolism, excretion and toxicity) in silico. Our findings support the rationale for further studies on EB5 derivatives and validation in animal models for prospective development as a new chemotherapeutic for clinical practice.
3. Discussion
Significant advancements in the understanding of the genetic and molecular landscape of the most prevalent pediatric tumors in recent years, following the identification of new potential therapeutic targets [
85,
86,
87], create new possibilities for treatment. However, no significant progress has been observed in the survival of patients with advanced or metastatic disease, and therapeutic protocols for the treatment of NR and RMS have not changed prominently since the 1980s [
4,
88,
89,
90]. Currently available treatment modalities, including surgical resection, radiation and standard chemotherapy, are ineffective and related to numerous heavy adverse effects. Subsequently, pediatric cancers still remain a significant clinical challenge [
5,
7,
14]. Therefore, multi-agent treatment and/or a multi-modality approach is urgently required.
Plant-derived chemicals are widespread in nature and are thus relatively readily available. Many of them have been proved to show a large spectrum of biological and pharmacological activities, including anti-bacterial, anti-viral, anti-fungal, anti-parasitic, anti-inflammatory [
91,
92], anti-oxidant and chemopreventive properties [
91,
92,
93,
94,
95,
96]. Consequently, significantly increasing interest in the use of natural substances as potential agents in the prevention and treatment of many human diseases, including cancer, is being observed nowadays [
97,
98,
99,
100]. Following this, the modification of phytochemicals with anti-tumor properties verified in cellular and animal models is a well-known method to obtain new compounds with improved pharmacokinetic parameters, activity and selectivity towards cancer cells for potential implementation in oncological treatment [
99,
101,
102].
Bet isolated from birch bark has been shown to demonstrate interesting chemopreventive and anti-cancer activities in vitro and in vivo, as previously reviewed in great detail [
16,
17]. The structure of Bet molecules makes several chemical modifications possible and thus it has been used as a starting compound for the synthesis of numerous new derivatives with potential applications in chemotherapy. The synthesis of mono- and di-acetylenic derivatives of Bet bearing acetyl substituents at the position of C-28 was reported for the first time in 2010 [
103]. In the present research, we showed the promising anti-cancer potential of two ASBDs carrying an acetylenic side chain at carbon C-28: 28-
O-propynoylbetulin (EB5) and 28-
O-propargyloxycarbonylbetulin (EB25/1) towards pediatric cancers cells in vitro. We found that both ASBDs prominently reduced cancer cells’ viability/survival and proliferation in a dose-dependent manner, also showing high selectivity for tumor cells measured by the selectivity index and relatively low or moderate activity against normal cells. More importantly, here, we demonstrated the considerably enhanced anti-survival and cytotoxic potential of ASBDs in comparison to IC
50 values of precursor compound Bet in SK-N-AS and TE671 cell lines showed previously [
18]. EB5, a derivative of Bet with a shorter alkynyl chain, showed evident stronger anti-survival, cytotoxic and anti-proliferative properties towards the analyzed pediatric cancer cells than EB25/1, a derivative carrying a longer alkynyl chain. The results from our in vitro study were highly consistent with the structure–activity relationships (SARs) in the literature, showing that the biological and pharmacological activity of ASBDs, including anti-tumor properties, was determined by differences in their chemical structure, especially their substituents. Several previous reports have showed that the introduction of an acetylenic side chain into the Bet structure resulted in the prominently increased anti-cancer activity of the derivatives when compared to a precursor molecule [
18,
25,
60,
61,
103,
104]. The carbon–carbon triple bond is considered to be one of the most crucial functional groups in organic and medicinal chemistry. The incorporation of alkyne moiety (acetylenic side chain, such as propynoyl in EB5) which contain a C-C triple bond was found essential for chemical, physical and biological properties of the molecule which determine its pharmacological profile. This simple modification at the position C-28 adjacent to the carbonyl group leads to formation of a very reactive chemical structure which may easily interact with other ligands and molecules [
104,
105,
106]. Therefore, alkyne substituents may enhance the affinity of the modified molecule to the nucleophilic amine and/or thiol groups in proteins within the cell membranes, following disturbances in their stability and integrity, and finally cause a decrease in cell viability and survival. Consequently, it is considered that alkyne groups at C-28 augment the affinity to the nucleophilic amine or thiol groups of proteins in the cellular membranes, affecting their integrity and stability and thus decreasing cell viability and survival [
60,
104,
106]. Similar to our study, other reports demonstrated the potent cytotoxicity and anti-proliferative activity of ASBDs towards cancer cells in vitro, including melanoma (G-361 cell line) [
104], human leukemia (CCRF/CEM) and murine leukemia (P388) [
25]. We have previously demonstrated that EB5 and EB25/1 significantly decreased the cell survival and proliferation of glioma cells (T98G and C6 cell lines), with IC
50 values being several-fold lower than those of TMZ when tested in vitro [
107]. In this study, we also showed several times (6.8 to 26) lower IC
50 values of EB5 for pediatric cancer cell lines than those observed for glioma cells, while the IC
50 doses of EB25/1 were quite similar in these different cellular models [
107]. Additionally, here, we found that EB5 showed increased cytotoxicity compared to conventional chemotherapeutic drug, CDDP, in analyzed cancer cell lines, and the combination of EB5 with CDDP at concentrations of IC
50 values considerably augmented anti-cancer activity in comparison to single treatment with EB5 or CDDP. Another study also showed that 28-acetylenic derivatives of Bet showed noteworthy anti-tumor effects towards human cancers of head and neck, ovarian, colon, lung, breast, thyroid and liposarcoma [
103]. Given the fact that the analyzed ASBDs demonstrated relatively weak cytotoxicity against human normal cells within the tested concentrations (0.5–25 µM) and the concentrations required to exert significant effects on viability/survival were multi-fold lower than these of TMZ, a chemotherapeutic drug considered for application in the therapy of NB [
8,
46,
47] and RMS [
48,
49,
50] patients, our research creates the opportunity for the development of new promising anti-tumor agents. Moreover, as CDDP is commonly used for the therapy of NB [
9,
10,
11,
51] and soft tissue malignancies, including RMS [
52,
53,
54], the combination of CDDP/EB5 supports the rationale for the usefulness of Bet derivatives combined with CDDP in the future development of novel therapeutic schedules. This kind of approach may potentially overcome frequently observed drug resistance and could result in the enhancement of anti-cancer activity and the reduction in toxicity and adverse effects [
10,
108] by a significant decrease in CDDP doses in clinical use. However, the pharmacological type of interactions between ASBDs and CDDP requires further analysis.
Next, we investigated the molecular mechanism underlying the ASBD-driven inhibition of pediatric cancer cells’ growth and proliferation. EB5 and other 28-acetylenic derivatives were also previously demonstrated as pro-apoptotic agents in ovarian A2780 and colon cancer SW480 [
103] and melanoma G-361 [
104] cell lines. We demonstrated the dose-dependent induction of apoptosis evidenced as upregulated levels of cleaved PARP and cleaved caspase 3, followed by the inhibition of signal transduction pathways commonly hyper-activated in many human tumors: Akt [
55,
56] and MAP kinases (Erk1/2 and p38) [
57,
58]. Involvement of the Akt pathway in the ASBD-mediated mechanism of action was also suggested by in silico studies. However, we cannot exclude other signaling pathways that may potentially contribute to anti-survival and anti-proliferative effects of ASBDs in analyzed cancer cell lines.
A growing number of evidence suggests that the early in silico evaluation of the ADMET profile and prediction of druglikeness of potential drug candidates play important roles in contemporary medicinal chemistry. Since the discovery and development of novel effective medicines and their subsequent implementation into clinical practice are extremely time- and cost-consuming processes, preliminary computational analysis would help in a significant decrease in failures in the clinical phases following an undesirable pharmacokinetic profile or unacceptable toxicity [
109,
110,
111]. Therefore, in this study, we also estimated the pharmacokinetic parameters and drug-like properties of ASBDs by the state-of-the-art in silico approach, implementing machine learning methods and algorithms.
Bet and its natural and synthetic derivatives, as many plant-derived secondary metabolites, are frequently insoluble or show very poor aqueous solubility [
31,
103,
112,
113]. Relatively low values of LogP determined for Bet are a consequence of two hydroxyl groups within its molecule, whereas the introduction of an acetyl moiety bearing additional carbon atoms into the Bet structure was followed by an increase in lipophilicity of ASBDs [
61]. In silico predictions, independently of the computational method used, revealed the considerable increase in LogP accompanied by diminished hydrosolubility (LogS) in the rank order Bet < EB5 < EB25/1. These results obtained by theoretical calculations are in agreement with previous experimental data from RT-TLC [
61]. Although the increasing lipophilicity leads to high fat solubility and consequently, improved lipid membranes permeation, the subsequent lowering of solubility in water may create several problems for the pharmacokinetic properties of the drugs, such as decreased absorption and distribution, and thus may result in limited in vivo administration and low bioavailability [
114,
115]. Nevertheless, this kind of obstacle could potentially be overcome by the complexation of ASBDs with hydrophilic carriers, such as
β-cyclodextrin [
113] and
γ-cyclodextrin derivatives [
31], by incorporation in nanoemulsion [
34] or encapsulation into hydrophilic vehicles, including liposomes [
34,
103]. On the other hand, our computational analysis showed the potential ability of ASBDs to pass through the BBB, since increasing lipophilicity may often significantly improve BBB permeation according to some literature reports [
116,
117]. Noteworthily, since we previously found significant anti-cancer effects of both EB5 and EB25/1 in glioma cells in vitro [
107], these properties of ASBDs may have potentially clinical-related relevance in the future if they are confirmed in a preclinical in vivo model. Our in silico analysis of ADMET parameters estimated good HIA of Bet and ASBDs; however, no permeability in the Caco-2 cell model and a lack of human oral bioavailability were predicted. In contrast, previously determined tPSA values for Bet and its acetylenic derivatives [
60] were less than 140, which may potentially suggest high oral bioavailability [
118,
119]. Several physicochemical parameters based on chemical structure, including molecular weight, lipophilicity, the number of H-BA and H-BD, tPSA, the number of atoms and rotatable bonds, were required for the evaluation of the probability of ASBDs being developed as medicines for humans. A useful method to predict a drug-like properties of the drug candidate is to determine its compliance with some druglikeness predictive guidelines. Bet and its derivative, EB5, were computationally demonstrated to follow two out of five drug-likeness guidelines: Lipinski and Veber, whereas for EB25/1, only Veber. According to the Lipinski “rule-of-five”, which is the most commonly used, and based on the observations that most orally administered medicines are rather small and moderately lipophilic molecules, an orally active drug candidate should not violate more than one of the following rules: MW less than 500 g/mol, MLOGP ≤ 4.15 (or LogP ≤ 5), number of H-bond acceptors (N or O atoms) less than 10, number of H-bond donors (NH or OH groups) ≤ 5 [
67,
120]. Here, we determined that Bet and derivative EB5 would fulfill the Lipinski “rule-of-five” with only one violation related to lipophilicity, while EB25/1 was found to violate two of these parameters since its MW is greater than 500 g/mol, and MLOGP ≥ 4.15 (LogP ≥ 5), and potentially, poor absorption or permeation is more likely for this Bet derivative, resulting in a higher probability of low human oral bioavailability. On the other hand, Bet and both ASBDs fulfilled the Veber druglikeness predictive guideline, which is only based on the number of rotatable bonds (≤10) and tPSA value (≤140) [
72]. Interestingly, other reports have shown that numerous well-known natural-derived medicinal agents, including macrocycles and cyclic peptides, may become human drugs in spite of the fact that they violate the Lipinski “rule-of-five” [
121,
122,
123].
In contrast to Bet’s subcellular localization of EB5 and EB25/1 predicted by ADMET, computational analysis may suggest potential diverse distribution and metabolic pathways between both ASBDs and their precursor molecule. The studied Bet derivatives, based on physicochemical properties, were demonstrated as neither substrates nor inhibitors of P-glycoprotein (P-gp) and subsequently could be prevented from undesirable efflux outside the cells, which is commonly involved in the multidrug resistance of numerous cancers to standard chemotherapeutics. P-gp, a transmembrane protein, is responsible for the transport of xenobiotics, including drugs out of the cells, and therefore can play a crucial role in the absorption and distribution of medicines [
124,
125]. Other protein families important for the transport and disposition of drugs are solute carrier (SLC) transporters: organic anion transporting polypeptides (OATPs), multidrug and toxin extrusion (MATE) and organic cation transporters (OCTs), which may affect the intracellular levels of several medications. OATPs are involved in the influx of endogenous and exogenous substances and mediate the hepatic uptake and distribution of endogenous agents and commonly used medicines, including standard chemotherapeutics [
111,
126,
127], whereas OCTs are poly-specific membrane transporters in the liver and kidney which contribute to the hepatic uptake of small, hydrophilic, positively charged molecules [
111,
128,
129,
130]. In contrast, MATEs have been described as proton/cation antiporters within plasma membrane and play important roles mainly in the efflux of xenobiotics, including the renal clearance of clinically used drugs [
128,
129,
130]. Our computational analysis of the ADMET profile predicted inhibitory effects of both Bet and ASBDs on OATP 1B1 and 1B3 isoforms, thus indicating their potential emerging role as candidates for pharmacological inhibitors of OATPs, since these transporting proteins were found to be significantly overexpressed in numerous human tumors suggesting the possible function of OATPs in cancerogenesis [
127]. Conversely, Bet and both EB5 and EB25/1 were determined as non-inhibitors of OCT2 and MATE1 transporters that could possibly suggest no ability to block OCT-mediated transport. Computational analyses have also found a potential inhibitory effect of Bet and ASBDs on the bile salt export pump (BSEP), suggesting possible involvement in liver and bile homeostasis, which could result in the increased concentration of bile salt in serum since BSEPs are the main transporters responsible for the secretion of bile acids and salts from hepatic cells [
131,
132].
The metabolism of chemical compounds has been regarded as one of the most crucial and difficult to predict parameters that should be taken into consideration during the design and development of novel drug candidates and ought to be carefully monitored in preclinical and clinical validations [
111,
133]. The cytochrome CYPP450 superfamily is a well-described large group of enzymes involved in the metabolic transformation of both endogenous and exogenous compounds [
134], including anti-cancer drugs or toxins, mainly in phase I reactions of oxidative biotransformations of many xenobiotics [
135]. Although, in silico analysis found ASBDs as non-inhibitors of most CYP450 isoforms, derivative EB25/1 was predicted as a substrate for CYP450 3A4 and CYP450 2C9, whereas Bet was predicted for CYP450 3A4. Essentially, neither EB5 nor EB25/1 were found to be carcinogenic/mutagenic or highly toxic agents, and they were classified as belonging to category III of toxicity according to the EPA [
78] (slightly toxic/irritating); however, EB25/1 was predicted as potentially hepatotoxic and corrosive to the eye.
The in silico prediction of potential biological targets within the cell or tissue for the analyzed ASBDs may also provide an insight into the molecular mechanisms underlying their anti-cancer activity. In this study, the proteins and signaling pathways which could be therapeutic targets for Bet derivatives were determined with the Balanced Substructure–Drug–Target Network-Based Inference (bSDTNBI) method using the NetInfer web server [
79,
80,
81]. Interestingly, among 10 targets of Bet as well as ASBDs with the highest scores, we found several members of the G protein-coupled receptor 1 (GPCRs) family and nuclear hormone receptor family. GPCRs are transmembrane proteins involved in the transduction of extracellular signals into cells, following the activation of G proteins and the modulation of numerous physiological and pathological processes, including tumorigenesis [
136,
137]. Nuclear hormone receptors are specialized transcription factors responsible for the regulation of specific gene expression by the promotion or repression of transcription [
138,
139]. Noteworthily, both these protein families are considered as the major targets for potential pharmacological intervention in several human diseases [
140,
141], which may suggest the future clinical relevance of the studied ASBDs. Furthermore, some target signaling cascades such as focal adhesion, PI3K-Akt, PPAR, ferroptosis and the p53 protein pathway predicted for both EB5 and EB25/1 in silico were found to be directly related to development and progression of human cancers. The targeting of focal adhesion proteins has been demonstrated to overcome the resistance of tumor cells to standard treatment modalities, such as chemo- and radiotherapy [
142,
143]. PI3K-Akt signaling is well known for the promotion of cancer survival, growth and proliferation, and its several pharmacological inhibitors are currently undergoing clinical trials or have been recently implemented into the clinical practice [
144,
145]. Importantly, computational prediction of the PI3K-Akt axis as one of the signal transduction pathways potentially affected by ASBDs was highly consistent with our findings from Western blot analysis, which showed reduced levels of phosphorylated Akt, following its decreased activity after treatment with both EB5 and EB25/1. The peroxisome proliferator-activated receptors (PPARs) are members of the ligand-inducible nuclear hormone receptor family involved in several processes related to metabolism and inflammation [
146,
147]. The regulation of the PPAR signaling cascade might be a possible new strategy to prevent carcinogenesis and progression [
148,
149]. Another signaling pathway potentially targeted by ASBDs was ferroptosis, a novel, recently discovered form of programmed cell death considered to be an interesting and promising player in the therapy of cancers [
150,
151]. Other pathways predicted as molecular targets for ASBDs which have not been directly linked to cancer so far, including complement and coagulation cascades, estrogen signaling, protein digestion and absorption, mineral absorption or hematopoietic cell lineage, need further investigation and confirmation in vitro and in vivo. Though it is difficult to speculate about the potential effects of ASBDs on the aforementioned signal transduction pathways based only on computational calculations, nevertheless, these findings may determine the directions for future molecular studies of both EB5 and EB25/1 and suggest new aspects to be analyzed in great detail.