The Effect of Oleanolic Acid and Its Four New Semisynthetic Derivatives on Human MeWo and A375 Melanoma Cell Lines

This study aimed to synthesize four new semisynthetic derivatives of natural oleanolic acid (OA) and, based on an analysis of their cytotoxic and anti-proliferative effects against human MeWo and A375 melanoma cell lines, select those with anti-cancer potential. We also screened the treatment time with the concentration of all four derivatives. We synthesized oxime 2 and performed its acylation with carboxylic acids into new derivatives 3a, 3b, 3c and 3d according to the methods previously described. Colorimetric MTT and SRB assays were used to measure the anti-proliferative and cytotoxic activity of OA and its derivatives 3a, 3b, 3c and 3d against melanoma cells. Selected concentrations of OA, the derivatives, and different time periods of incubation were used in the study. The data were analyzed statistically. The present results revealed the possible anti-proliferative and cytotoxic potential of two selected OA derivatives 3a and 3b, on A375 and MeWo melanoma cells, especially at concentrations of 50 μM and 100 μM at 48 h of incubation (p < 0.05). Further studies will be necessary to analyze the proapoptotic and anti-cancer activities of 3a and 3b against skin and other cancer cells. The bromoacetoxyimine derivative (3b) of OA morpholide turned out to be the most effective against the tested cancer cells.


Introduction
Human skin is the organ most exposed to environmentally damaging and mutagenic factors, which may result in cancer formation. The most common skin cancers are basal cell carcinoma and squamous cell carcinoma [1][2][3], although malignant melanoma is considered the most deadly, being responsible for 90% of deaths associated with cutaneous tumors [4]. Melanoma is caused by the abnormal proliferation of melanocytes [5]. These pigmentproducing cells are present in the basal layer of the epidermis. Melanocytes are derived from a neural crest origin and produce many signaling molecules and other bioactive factors that promote migration and metastasis after malignant transformation [5,6]. Most types of malignant melanoma are believed to be caused by sun (UV) exposure without protection, although gender (men), age (above 65 years old), family history, genetic predispositions, indoor tanning, moles or nevi, and obesity may contribute to this disease [4][5][6]. In recent years, there has been an increase in malignant melanoma in white men over 60 years of age in Sweden and northern Europe [4,6,7]. An increase in melanoma incidence was also observed in all age groups in other parts of Europe [4,7], and this situation has not stabilized [6,7]. Primary melanoma usually appears in a cutaneous location, although this tumor metastasizes quickly [8,9]. Malignant melanoma can have several systemic consequences. After it spreads to other areas of the body, such as the lungs, liver, brain, and bones, metastatic melanoma causes damage to these organs and affects their normal

Synthesis of Cytotoxic Agents
To explore the effects of substituents within the acyloxyimino group on cytotoxic activity, the C-3 hydroxyl group was converted into oxo, and then into hydroxyimino and acyloxyimino groups. As previously shown, the transformation of the C-17 carboxyl group into morpholide moiety leads to an improvement of cytotoxic activity of OA derivatives, which is why the second transformation of the OA structure was the introduction of a morpholide ring instead of hydroxyl within the -COOH function [30].  Figure 1 shows that OA (1) was the starting material for performing our syntheses [31]. The obtained oxime (2) was acylated by using a procedure known from the literature data [31]. As a result, four acylated oximes of OA morpholide (3a-3d) were obtained, with the carboxylic group at the C-17 position transformed into an amide system. The structures of the obtained acylated oximes were confirmed with the spectral data ( 1 H NMR, 13 C NMR, DEPT).
To explore the effects of substituents within the acyloxyimino group on cytotoxic ac tivity, the C-3 hydroxyl group was converted into oxo, and then into hydroxyimino and acyloxyimino groups. As previously shown, the transformation of the C-17 carboxy group into morpholide moiety leads to an improvement of cytotoxic activity of OA deriv atives, which is why the second transformation of the OA structure was the introduction of a morpholide ring instead of hydroxyl within the -COOH function [30]. Figure 1 shows that OA (1) was the starting material for performing our synthese [31]. The obtained oxime (2) was acylated by using a procedure known from the literatur data [31]. As a result, four acylated oximes of OA morpholide (3a-3d) were obtained, with the carboxylic group at the C-17 position transformed into an amide system. The struc tures of the obtained acylated oximes were confirmed with the spectral data ( 1 H NMR, 13 C NMR, DEPT).  (2), acylated oximes of OA morpholide (3a-3d). i: Jones reagent, acetone, rt.; ii: (COCl2), rt.; morpholine, benzene, rt.; ii NH2OH x HCl, CH3COONa, ethanol, reflux, iv: dioxane, DCC, (Ar)RCOOH, rt.

Effects of OA and Its Derivatives on the Viability of Human MeWo Cells-MTT and SRB Assays
The effects of selected OA concentrations and its derivatives on the viability of the human MeWo melanoma cells were evaluated following a treatment period of 6 h, 24 h and 48 h using an MTT assay ( Figure 2).
OA and its derivatives 3a, 3c, and 3d at each concentration did not exert any cytotoxic activity after 6 h of incubation. Derivative 3b at 100 µM reduced MeWo cell viability to 72.2% (Figure 2A).
After 24 h, derivative 3a at the highest tested concentration of 100 µM significantly decreased cell viability to 69.1%, while derivative 3b significantly decreased cell viability to 61.5% and 39.2% at 50 and 100 µM, respectively. We did not observe significant cytotoxic effects for the remaining tested derivatives after 24 h of incubation ( Figure 2B).
In the SRB test (Figure 3), no statistically significant reduction in human MeWo cell viability was demonstrated after 6 h of treatment with different concentrations of OA and its derivatives ( Figure 3A). OA and its derivatives 3a, 3c, and 3d at each concentration did not exert any cytotoxic activity after 6 h of incubation. Derivative 3b at 100 µM reduced MeWo cell viability to 72.2% (Figure 2A).
After 24 h, derivative 3a at the highest tested concentration of 100 µM significantly decreased cell viability to 69.1%, while derivative 3b significantly decreased cell viability to 61.5% and 39.2% at 50 and 100 µM, respectively. We did not observe significant cytotoxic effects for the remaining tested derivatives after 24 h of incubation ( Figure 2B).
In the SRB test ( Figure 3), no statistically significant reduction in human MeWo cell viability was demonstrated after 6 h of treatment with different concentrations of OA and its derivatives ( Figure 3A).   Descriptive data were presented as mean ± SD. Statistically significant differences versus control cells were marked with an asterisk (* p ≤ 0.05). The horizontal bold line represents the viability of control (untreated) cells.

Effects of OA and Its Derivatives on Viability of Human A375 Cells-MTT and SRB Assays
The effects of OA and its derivatives on the viability of the human A375 melanoma cells were evaluated following a treatment period of 6 h, 24 h and 48 h using MTT and SRB assays (Figures 4 and 5). of 100 µM, it significantly reduced viability to 51.1% ( Figure 3C).

Effects of OA and Its Derivatives on Viability of Human A375 Cells-MTT and SRB Assays
The effects of OA and its derivatives on the viability of the human A375 melanoma cells were evaluated following a treatment period of 6h, 24h and 48h using MTT and SRB assays (Figures 4 and 5).   The results of the MTT assay (the metabolic activity test) on the viability of A375 cells incubated with triterpenes OA, 3a, 3b, 3c, or 3d for (A) 6 h, (B) 24 h or (C) 48 h. Descriptive data were presented as mean ± SD. Statistically significant differences versus control cells were marked with an asterisk (* p ≤ 0.05). The horizontal bold line represents the viability of control (untreated) cells.  No statistically significant reduction in human A375 cell viability was demonstrated after 6 h treatment with different concentrations of triterpenes ( Figure 4A).
The results observed in the SRB were varied. After 6 h of treatment, derivative 3a significantly reduced the viability of A375 melanoma cells under the following conditions: at 5 µM to 82.5% and at 100 µM to 80.9%. Derivative 3c at 0.5 and 1 µM decreased cell viability to 85.2% and 83.5%, respectively. No cytotoxic effect was observed at higher concentrations of 3c ( Figure 5A). Derivative 3d at 5 and 100 µM µM decreased cell viability to 88.7% and 86.0%, respectively. A similar effect was observed for OA, which at concentrations of 5 and 100 µM decreased cell viability to 82.6% and 82.4%, respectively ( Figure 5A).

Discussion
Chemotherapy, targeted therapy and immune checkpoint inhibitor therapy are more effective after the surgical excision of the primary tumor at any stage of cutaneous melanoma [5,12]. Additionally, synthetic drugs usually act equally on normal and tumor cells, which may cause more harm than selective drugs [16,17]. The combination of therapeutic efficiency with the lack of toxic activity makes biologically active compounds, such as triterpenes, an attractive alternative to conventional treatment [16,17]. One of the best-known representatives of pentacyclic triterpenes is OA, which exhibits anti-cancer effects on human carcinoma cell lines. OA participates in blocking the migration and invasion of thyroid cancer cells and hepatocellular carcinoma cells [32,33]. Moreover, OA inhibits rectal cells proliferation [34] and induces autophagy and apoptosis in colon cancer cells and hepatoma cells [35,36]. However, studies have confirmed that OA is characterized by poor solubility in water, which limits its use in pharmacotherapy [25,37]; however, it can be successfully modified to improve its broad activity and bioavailability. Several OA derivatives, acylated in various manners in the 3-hydroxyimino group, have been described in numerous research articles. They all showed higher anti-cancer activity than the starting OA. Here, we prepared four new OA derivatives, which exhibited better anti-proliferative and cytotoxic effects on human melanoma cell lines; furthermore, possibly, due to the presence of a morpholine moiety at the C-28 position, they have more advantageous characteristics than other similar derivatives. Here, as a result of our research, the effect of the acyl group's structure at the C-3 position of the 3-hydroxyimino OA derivatives on the anti-proliferative activity of the obtained new compounds was determined.
We tested the OA and its derivatives to determine its cytotoxic effects against human MeWo and A375 melanoma cell lines with an SRB assay, and the anti-proliferative properties of substances were measured with an MTT assay. The cells were incubated with triterpenes for 6 h, 24 h and 48 h.
The results of the MTT assay showed that the 3b derivative exerts a significant doseand time-dependent anti-proliferative effect in both melanoma cell lines. Analyzing the six-hour incubation time, in the case of MeWo cells, only the 3b derivative at a concentration of 100 µM showed a significant anti-proliferative effect on the tested cells. Due to the longer incubation time, the 3b derivative already significantly inhibited MeWo cell proliferation at a minimum concentration of 10 µM. The SRB assay used at the same incubation times and with the same concentrations of the 3b substance showed a slightly weaker cytotoxic effect on MeWo cells. In this case, a statistically significant reduction in cell viability was demonstrated for a concentration of 50 µM after 48 h incubation.
The second compound demonstrating a significant anti-proliferative effect on MeWo cells was the 3a derivative. The MTT assay showed its statistically significant inhibition of cell viability after 48 h of incubation and at a minimum concentration of 25 µM. In the case of the SRB assay, the cytotoxicity of the 3a derivative was also demonstrated after 48 h of incubation, but at a minimum concentration of 50 µM. Derivatives 3c and 3d exerted cytotoxic activity after 48 h of incubation and at concentrations of 50 µM and 100 µM only in the MTT assay. We demonstrated that OA did not induce any changes in the MeWo cell viability regardless of the tested concentrations.
Ours is the first study to evaluate the cytotoxicity of OA derivatives on MeWo cell lines; therefore, our obtained data cannot be compared with other studies. Welch et al. [30] reported that the MeWo melanoma cell line is characterized by a moderate metastatic potential compared with other melanoma cell lines, for example A375. It has been shown that cells of the MeWo line have exhibited prediction to extrapulmonary metastases.
OA and its derivatives were also tested on the A375 melanoma cell line. Again, the most promising results in the MTT assay were obtained for the 3a and 3b derivatives. The application of the 50 µM sample concentration with 24 h incubation, and the cell treatment of a minimum of 10 µM for 3b or 25 µM for 3a within 48 h, induced significant decreases in cell viability. Cytotoxic effects also occurred for the 3b derivative at a minimum of 10 µM and 24 h or 48 h of incubation in the SRB assay, whereas derivative 3a demonstrated a cytotoxic effect after 6 h of incubation at a concentration of 100 µM or at 50 µM and 48 h of incubation in the SRB assay. Derivatives 3c and 3d showed anti-proliferative activity at a concentration of 50 µM and 48 h of incubation in the MTT assay. Only the 3d derivative significantly decreased cell viability at 100 µM and 6 h of incubation or at 50 µM and 48 h of incubation. Our findings confirmed that 3-hydroxyimine derivatives of OA morpholides show anti-cancer potential higher than the starting and comparative unmodified OA. Moreover, the acylation of the hydroxyimine group may have a beneficial cytotoxic effect in the used cell lines. Comparing the effectiveness of new compounds with various types of acyl substituents, we concluded that the highest efficiency was shown by products with an aliphatic substituent, especially with an electron-withdrawing substituent.
In the case of the A375 cell line, OA exerts a cytotoxic effect at a concentration of 100 µM and 24 h or 48 h of incubation (MTT assay). However, the SRB assay did not show a significant inhibition of cell viability after the application of each OA concentration. The results obtained by Mioc et al. [23] confirmed that OA at 50 µM and a treatment period of 24 h significantly decreased A375 melanoma cell viability to 74.8%. They also tested benzotriazole ester of OA, which showed a better dose-dependent reduction in cell viability against A375 cells and no cytotoxic effect against healthy human keratinocytes. Additionally, Oprean et al. [22] demonstrated that OA exerts a significant cytotoxic effect on A375 melanoma cells at 50 µM and 48 h of incubation (78% cell viability), although this effect was determined to be low to moderate compared with the cytotoxic activity of ursolic acid (UA). Research conducted on the SK-MEL-2 melanoma cell line demonstrated that UA exerted a significant dose-dependent anti-proliferative effect in vitro compared with OA.
Further testing the two compounds as a mixture is needed to reveal the possible synergic or additive effects on blood vessels and tumor cells [18]. Isakovic-Vidovic et al. [24] reported the promising inhibitory effects of OA mixtures with betulinic acid and UA on WM-266-4 metastatic melanoma cells proliferation activity.
The present results reveal the possible anti-proliferative and cytotoxic potential of two selected OA derivatives, namely 3a and 3b, on A375 and MeWo melanoma cells. Further studies will be necessary to analyze the pro-apoptotic and anti-cancer activities of the 3a and 3b derivatives against kin and other cancer cells. The bromoacetoxyimine derivative of OA morpholide was the most effective against the tested cancer cells.
In summary, we synthesized four new OA derivatives and determined that two of them, 3a and 3b, showed significant anti-cancer activity against the MeWo and A375 melanoma cell lines. This indicates that alkyl derivatives are preferable to aryl ones in the present experiment. In addition, we determined that the most effective concentrations for 3a are 50 and 100 µM at 48 h for both tested cell lines, while for derivative 3b it is 100 µM with 24 h of incubation and 50 µM, and 100 µM with 48 h of incubation for both tested cell lines.
In the future, we are planning to study appropriately selected compounds on other melanoma cells and normal cells, including fibroblasts and keratinocytes, because melanoma mainly proliferates in the skin.

General Synthesis Procedure for Derivatives 3a, 3b, 3c and 3d
• General information All commercially available solvents and reagents used in our experiments were graded "pure for analysis" (Chempur or Sigma-Aldrich). The solvents were dried according to the usual procedures. Products were purified by column chromatography using 70-230 mesh silica gel (Merck). TLC analysis was performed with the application of benzene and ethyl acetate in given volume ratios. Melting points were measured with a Büchi apparatus in an open capillary and are uncorrected. Elemental analyses (C, H, N) were performed with a Perkin-Elmer 2400 CHN analyzer. 1 H and 13 C NMR spectra were recorded on a Varian Gemini 300 VT spectrometer for frequencies of 300 MHz and 75 MHz, respectively. TMS (δ = 0 ppm) was used as an internal standard. The multiplicity are reported as follows: s = singlet, d = doublet, t = triplet, qu = quartet, m = multiplet, dd = doublet doublets, dt = doublet triplets, br/s = broad singlet. ESI-MS spectra were recorded on a QTOF (5600+, AB Sciex) spectrometer.

•
Synthesis of oximes 2 Synthesis of oxime 2 was performed according to methods known from literature data [38]. Physical and spectral data were in agreement with those from literature data [38].

•
Acylation of oxime 2 with carboxylic acids Acylation of oxime 2 with carboxylic acids was performed according to methods known from literature data [38] starting from 1.0 mmol of each oxime.

Cell Culture
Two certified human malignant melanoma cell lines: A375 (ATCC ® CRL-1619™) and MeWo (ATCC ® HTB-65™) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultured in a complete growth medium that contained Dulbecco's Modified Eagle Medium without phenol red (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with addition of stabilized 1% antibiotic antimycotic solution containing 25 µg/mL of amphotericin B, 10,000 units/mL of penicillin, and 10 mg/mL of streptomycin (Sigma-Aldrich, St. Luis, MO, USA). Medium was renewed every 3 days. Cells were cultured in a CEL-CULTURE ® CCL-170B-8 CO 2 incubator (Esco, Singapore) at 37 • C in 95% humidified air with 5% CO 2 . Cells were harvested from cell culture T-75 flasks (Eppendorf AG, Hamburg, Germany) with TrypLE™ Express (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), stained with 0.4% trypan blue solution and counted with Countess™ Automated Cell Counter (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). For the experiments, the cells were grown on 96-well tissue-culture-treated microplates (Eppendorf AG, Hamburg, Germany), seeded at 15,000 cells per well, and incubated overnight to allow attachment, followed by treatment with OA and its derivatives for 6, 24 and 48 h at a concentration range of 0.75-100 µM. Control cells were incubated with 0.1% dimethyl sulfoxide (DMSO) solvent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in a complete growth medium.

Evaluation of Cell Metabolic Activity of Cells Treated with OA and Its Derivatives with the MTT Assay
After the treatment time specified in the experiment conditions, the post-culture medium was discarded, cells were rinsed with sterile phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) solution, and a freshly prepared 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in complete growth medium (MTT reagent; Sigma-Aldrich, St. Luis, MO, USA) was added to the culture. Plates were incubated for 3 h in the CO 2 incubator under the above-mentioned conditions. Subsequently, the MTT reagent was gently decanted, and the formed formazan crystals were dissolved in DMSO (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The absorbance was measured with Infinite ® M200 spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland) at λ = 540 nm. The experiments were conducted in triplicate, utilizing cells from different cell passages.

Evaluation of the Cells Number after Exposure to OA and Its Derivatives with the Sulforhodamine B (SRB) Assay
After the treatment time specified in the experimental conditions, cells were fixed with trichloroacetic acid (TCA; Sigma-Aldrich, St. Luis, MO, USA) at a final concentration of 12.5% and incubated for 1 h at 4 • C, followed by gentle rinsing with cold water and drying. Subsequently, a freshly prepared solution of 0.04% SRB (Sigma-Aldrich, St. Luis, MO, USA) in 1% acetic acid (Avantor Performance Materials Poland, Gliwice, Poland) was added and left at room temperature for 30 min. The unbound dye was removed using 1% acetic acid. The protein-bound SRB was solubilized in 10 mM Tris base solution (BioShop, Burlington, Ontario, Canada), pH 10.5. The absorbance, proportional to the protein content, was measured using the Infinite ® M200 spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland) at λ = 520 nm. The experiments were performed in triplicate, utilizing cells from different cell passages.

Statistical Analysis
Descriptive data were shown as a mean and a standard deviation (± SD). Distribution of the data was tested with the Shapiro-Wilk normality test, and the homogeneity of variances were analyzed by Leven's test. One-way analysis of variance ANOVA was used for multiple comparison procedure, and the post-hoc Tukey test was used for evaluation of differences between control and study groups. Values with p ≤ 0.05 were considered to be statistically significant. Data were analyzed using MS Excel 2016 (Microsoft Co, USA) and Statistica v.13.3 (Tibco Software Inc., Palo Alto, CA, USA).