Isolation of the Lanostane Triterpenes Pholiols L–S from Pholiota populnea and Evaluation of Their Antiproliferative and Cytotoxic Activities

Pholiols L-S (1–8), eight undescribed triterpenes were isolated from the sporocarps of the mushroom Pholiota populnea. Various chromatographic techniques, such as open column chromatography, flash chromatography, gel filtration, preparative thin layer chromatography, and HPLC, were applied to purify the compounds. The structure elucidation was carried out by spectroscopic analysis, including 1D (1H NMR and 13C JMOD) and 2D NMR (1H-1H COSY, HSQC, HMBC and NOESY) and HRESIMS experiments. The isolated compounds had lanostane (1–7) or trinorlanostane (8) skeletons; all of them were substituted with 3-hydroxy-3-methylglutaroyl group or its 6-methyl ester. Five compounds (1, 2, 4, 6, and 8) were investigated for their antiproliferative and cytotoxic activity in vitro by MTT assay on breast cancer (MCF-7), human colon adenocarcinoma (sensitive Colo 205, and resistant Colo 320), non-small cell lung cancer (A549), and human embryonic lung fibroblast (MRC-5) cell lines. Pholiols M (2) and O (4) showed antiproliferative activity against the MCF-7 cell line with IC50 of 2.48 and 9.95 µM, respectively. These compounds displayed tumor cell selectivity on MCF-7 cells with SI values of >40 (2) and 4.3 (4), but they did not show a cytotoxic effect, proving their action exclusively on tumor cell proliferation. Pholiols L (1) and Q (8) were found to have selective cytotoxicity on drug resistant cells in comparison to their effects on Colo320 and Colo205 cells [relative resistance values 0.84 (1) and 0.62 (8)].


Introduction
For many decades, mushrooms have been utilized because of their beneficial nutritional and medicinal values, including immunomodulatory and antitumor activities. Fungal metabolites can have a protective effect by stimulating the immune response and activating immune cells, macrophages, T cells, and natural killer cells. Furthermore, bioactive compounds of mushrooms have shown cytotoxicity, antiproliferative activity, apoptosisinducing properties, and have been shown to act as cancer cell metastases inhibitory agents. Certain dietary mushrooms (e.g., Agaricus bisporus) were reported to contain anticarcinogenic metabolites which alter the aromatase enzyme activity. The active ingredients responsible for the immunomodulatory and antitumor activities of mushrooms include polysaccharides, polysaccharide-protein complexes, lectins, amino acids, polyphenols, sterols, and other terpenoids [1][2][3].
Polysaccharides from Pholiota adiposa, especially SPAP2-1, showed strong interference of the cell cycle of HeLa cells and induced apoptosis [4]. A protein purified from the edible fungus Pholiota nameko (PNAP) shows promise for the treatment of breast cancer, as it induced apoptosis of human breast adenocarcinoma MCF-7 cells in vivo and modulated cytokine secretion in mice bearing MCF-7 xenografts [5]. The glucan polysaccharides schizophyllan, derived from Schizophyllum commune, and lentinan, produced by Lentinula edodes, were investigated in clinical trials, and a better overall survival of patients with head and neck cancer and gastric and colorectal carcinomas, respectively, were found [6].
Ganoderma lucidum polysaccharides and triterpenoids were found to be potent inhibitors of tumor growth in vitro and in vivo [7]. Moreover, extracts of G. lucidum and G. tsugae were able to inhibit the growth of colorectal cancer cells in vitro [8]. Ergosterol, the main sterol of mushroom species, is able to induce apoptosis in leukemia cells and inhibits tumor-induced angiogenesis [9]. Hypholoma lateritium (Strophariaceae) was reported to contain lanostane-type triterpenoids, sublateriols A-C, and fasciculol A-C, some of them exhibited cytotoxicity against human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) in vitro [10].
In our previous study, Pholiota populnea was investigated for antitumor compounds, and lanostane diesters, named pholiols A-D, ergosterol, 3β-hydroxyergosta-7,22-diene, and a polyhydroxy-squalene derivative were isolated. Ergosterol was found to show cytotoxic activity against sensitive (Colo 205) and resistant colon adenocarcinoma cells (Colo 320) with IC 50 values of 4.9 and 6.5 µM, respectively. The p-glycoprotein (ABCB1) efflux pump inhibitory activity of the compounds was also tested against resistant Colo 320 cells and inhibitory activity was found for pholiols A and B and the squalene derivative. In addition, the drug interactions of triterpenes with doxorubicin were also studied by the checkerboard method on Colo 320 cells, and it was observed that pholiols B and D interacted in a synergistic manner, while the acyclic triterpene acted in a very strong synergistic manner [11]. Further mycochemical investigations led to the isolation of pholiols E-K and (+)-clavaric acid, some of the triterpenes exerted moderate COX-2 and 5-LOX inhibitory activities. Pholiol F (IC 50 194.5 µM against 5-LOX, and 439.8 µM against COX-2) was found to be the most effective among the investigated compounds [12].
Compound 5 (pholiol p) was obtained as a white amorphous solid with an optical rotation of [α] 23 −1.6 (c 0.05, MeOH). Its molecular formula was deduced to be C 39 H 56 O 10 from HRESIMS sodium adduct ion peak at m/z 683.3767 [M + Na] + (calcd for C 37 H 56 O 10 Na + 683.3766). Comprehensive analysis of 1 H NMR, 13 C JMOD and 2D NMR data of 4 and 5 indicated the presence of the same 2,3,25-trihydroxy-7,11,24-triketo-triterpene core and MeHMG esterification at C-2 as discussed at compound 4, with the exception of the acetoxy group at C-3, which was replaced by a hydroxy group. This was clearly shown by the H-3 signal at δ H 3.28 d (J = 11.0 Hz) for 5, instead of H-3 at δ H 3.28 d (J = 11.0 Hz) for 4 (Tables 1 and 2

Evaluation of the Antiproliferative and Cytotoxic Activity of the Isolated Compounds
The compounds isolated in good yield, such pholiols L, M, O, Q, and S (1, 2, 4, 6, and 8), were investigated for their antiproliferative activity in vitro by MTT assay in human colon adenocarcinoma cell lines, both sensitive (Colo 205) and resistant (Colo 320) ones, hormone-responsive breast cancer (MCF-7), human non-small cell lung cancer (A549), and human embryonic lung fibroblast cell line (MRC-5). The anticancer drug doxorubicin was used as a reference agent. The results, obtained as the concentration of the compound that produced half of the inhibition (IC 50 ), are shown in Table 3. The highest antiproliferative effects were observed for pholiols M (2) and O (4) against the MCF-7 cell line, with IC 50 values of 2.48 and 9.95 µM, respectively. These compounds were also potent against Colo 205 cells, but to a lower extent [IC 50 23.91 (2), and 23.30 µM (4)], while pholiol L (1) was moderately active on the MRC-7 cell line (IC 50 21.74 µM). Structurally, compounds 2 and 4 are lanostane triterpenes with 2,3-diester-7-on-8 (9)-en functionalities, with a further 11-keto (4) or 12-hydroxy group (2). All other compounds exhibited weaker activity on the viability of the treated cancer cells, displaying IC 50 values ranging from 28.07 to 89.84 µM. Compounds 1, 2, 4, 6, and 8 were tested for their antiproliferative activity in the noncancerous human embryonic lung fibroblast cell line as well, and on the basis of these results, selectivity indexes (SI) were calculated. The SI values of pholiol M (2) > 40 and 4.18 indicated its strong and moderate tumor cell selectivity regarding the MCF-7 and Colo 205 cell lines, respectively, whereas SI values of 4.3 and 1.8 of pholiol O (4) indicated this compound to be moderately or slightly selective towards Colo 205 cells [14]. Compounds 1, 6, and 8 did not display selectivity to cancerous cell lines over normal cells.
The direct cytotoxic effects of the compounds were determined similarly by MTT assay on the Colo 205, Colo 320, MCF-7, A549, and MRC-5 cell lines, using a higher cell population and a shorter exposure than in the antiproliferative test. Under these conditions, the direct killing effect can be measured instead of the cell growth inhibitory activity. Significant difference between the antiproliferative and cytotoxic effects displayed selectivity for the growing cell population without directly killing the exposed cells. Such a result was exhibited by pholiols M (2) and O (4), for which, no cytotoxic effect could be detected on MCF-7 cells (IC 50 > 100 µM), proving their action exclusively on tumor cell proliferation. Considering all other compounds and cell lines, moderate cytotoxic activities were measured with IC 50 from 31.52 to over 100 µM (Table 4). Table 3. Antiproliferative activity (IC 50 µM) of pholiols L, M, O, Q, and S (1, 2, 4, 6, and 8) in human sensitive (Colo 205) and resistant (Colo 320) colon adenocarcinoma cells. and normal embryonal fibroblast (MRC-5) cell line.

Compd.
Colo  Comparison of the cytotoxic activities on drug-resistant (Colo 320) and sensitive (Colo 205) cells allowed for the detection of relative resistance (RR), which was calculated as the ratio of the IC 50 value in the resistant and in sensitive cancer cell lines. Compounds with RR < 1 show selectivity against resistant cells, whereas RR ≤ 0.5 means that the compounds have a collateral sensitivity (CS) effect [15]. The calculated RR values of the tested compounds showed selectivity against the resistant Colo 320 cells of pholiols L (1) (RR 0.84) and Q (8) (RR 0.62) ( Table 4).

Spectroscopic and Physical Characteristic of Compounds
IC 50 = OD sample − OD medium control OD cell control − OD medium control × 100 (1) Assay for Cytotoxic Effect: The effects of increasing concentrations of the compounds on cell growth were tested in 96-well flat-bottomed microtiter plates, as described for the antiproliferative assay, using 1 × 10 4 cells/well. The culture plates were incubated at 37 • C for 24 h; at the end of the incubation period, 20 µL of MTT solution (from a 5 mg/mL stock solution) was added to each well. After incubation at 37 • C for 4 h, 100 µL of sodium dodecyl sulfate (SDS) solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were further incubated at 37 • C overnight. Cell growth was determined by measuring the optical density (OD) at 540 nm (ref 630 nm) with a Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). Inhibition of cell growth was expressed as IC 50 (Table 4).
Sample preparation: The compounds were dissolved in DMSO to achieve the final concentration of 5 mM. The starting concentration of the compounds was 100 µM and 2-fold serial dilutions were prepared for the antiproliferative and cytotoxic assays. The following concentrations were used: 100-50-25-12.5-6.25-3.125-1.56-0.78-0.39-0.195 µM.