A Saprophytic Fungus Tubeufia rubra Produces Novel Rubracin D and E Reversing Multidrug Resistance in Cancer Cells

Two new (1, 2 viz Rubracin D and E) and sixteen known Glyceroglycolipids (3–18) in the saprophytic fungus Tubeufia rubra (PF02-2) from decaying wood in freshwater habitat were isolated and identified. Their chemical structures were elucidated via means of the extensive spectroscopic analyses of NMR, HR-ESI-MS and UV spectra, as well as comparison with literature data. The new compounds were assayed for the reversal activity of multidrug resistance (MDR) on MCF-7/ADM, K562/ADM and A549/ADM cell lines, and both compounds 1 and 2 reversed MDR in the three resistant cancer cell lines with concentration dependence. In the assay on K562/ADM, both new compounds had been proved to have remarkable MDR reversal effects, which were higher than those of the positive control viz Verapamil (Vrp). Meanwhile, in the assay on A549/ADM, compound 1 displayed significant MDR reversal effects, which were also higher than those of Vrp at certain concentrations. Furthermore, the Western blot assay proved that both new compounds reversed the MDR in the resistant cancer cell line viz MCF-7/ADM by inhibiting the overexpression of P-glycoprotein. This is the first report that the Glyceroglycolipids isolated firstly from the fungal genus Tubeufia reversed MDR in resistant cancer cells.


Introduction
Cancer is still a leading cause of death worldwide [1,2]. Multidrug resistance (MDR) in cancer cells is a key factor in malignancies treatment with chemotherapeutic drugs [3]. P-glycoprotein (P-gp), a 170-kDa transmembrane protein, plays an important role in the process of MDR with its overexpression. Searching for P-gp inhibitors that can be combined with anticancer drug therapies is one of the most efficient strategies to reverse cancer MDR [4]. Natural products are important bioactive agents for developing a new clinic drug because of their diverse structures and potential biological activities [5][6][7][8][9]. A large number of natural products have reported the potential to inhibit the overexpression of P-gp resulting in reversing MDR activity in tumors [10][11][12] with structures involved in alkaloids [13], flavonoids [14], lignans [15,16] and terpenoids [17,18].
To date, many bioactive secondary metabolites were reported from the microorganism, in 1993, Stierle et al. [19] found that the strain Taxomyces andreanae isolated from the phloem (inner bark) of Taxus brevifolia had the ability to produce taxol, and numerous natural products with complicated structure and novel activity [20][21][22]. For example, the esters of orsellinic acid derivatives globosumones A-C isolated from Chaetomium globosum exhibited significant cytotoxic activity against four cancer cell lines NCI-H460, MCF-7, SF-268, and WI-38 [23]. Two novel xanthone dimers phomoxanthones A and B obtained the endophytic fungus Phomopsis sp. BCC 1323 showed antimalarial and antitubercular activities [24]. A new chromone aspergilluone A produced by a marine sponge-associated fungus Aspergillus sp. LS57 displayed antimicrobial activity against Staphylococcus aureus and Bacillus subtilis [25]. The secondary metabolites from Helicomyces sp. No.19353 demonstrated significant antidiabetic activity [26]. Therefore, it is important to find active substances in microbial fermentation broth.
Our previous studies indicated that the crude ethyl acetate extract of the fermentation broth from the strain Tubeufia rubra (PF02-2) showed significant reversal activity of MDR in MCF-7/ADM cell line via inhibiting the overexpression of P-gp [27]. A novel nitrogencontaining compound Rubracin A with reversed MDR activity was isolated from the strain Tubeufia rubra (PF02-2) [28], while the other active compounds awaited clarification. In this study, the saprophytic fungus PF02-2 of the decaying wood in a freshwater habitat and the secondary metabolites from its fermented culture were further investigated. Herein, the isolation, structure elucidation and bio-activity assay of another new class of compounds from the crude ethyl acetate extract of PF02-2 were described in detail.

General Experimental Procedures
HR-ESI-MS was performed on a Waters Xevo G2 QTOF mass spectrometer. IR spectra were recorded with NICOLET iS10. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. Optical rotations were measured on a JASCO-20C digital polarimeter. Then, 1D NMR and 2D NMR were performed on Bruker DRX-500, or AVANCE III-600 spectrometers with TMS as an internal standard. Silica gel and Sephadex LH-20 were used for column chromatography (CC). Medium-pressure liquid chromatography (MPLC) was performed on a Lisui EZ Purify III System including pump manager P03, detector modules P02, and fraction collector P01 and columns packed with RP-18 gel. Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.

Fungus Materials
The fungal strain (PF02-2) was isolated from decaying wood in a freshwater habitat, which was collected in Fangchenggang City, Guangxi Province, China, in May 2016. This strain was reported as a new species viz Tubeufia rubra by Lu et al. [29]. The strain is preserved at the Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University (GZCC 16038) and China Center for Type Culture Collection (CCTCC NO. M2019957), Wuhan University.

Fermentation, Extraction and Isolation
A stock culture of the strain was maintained at 4 • C on PDA medium (Solarbio). The stock culture was inoculated into potato dextrose agar medium (PDA) at 28 • C for 17 days. A flask of 250 mL containing 100 mL of seed medium (PDB) was inoculated with six agar chunks with an area of 1 × 1 cm 2 from a well-grown culture of PF02-2 and shaken on a rotary shaker at 28 • C and 180 rpm for 3 days according to the preliminary experimental results. The seed cultures were then used to inoculate the production-scale fermentation. Each seed culture (2 mL/piece) was transferred into a flask of 1 L containing 200 g oat and 150 mL distilled water and fermented at 28 • C for 105 days. The whole fermented cultures (30 kg) were extracted three times with EtOAc at room temperature for 48 h on a rotary shaker (50 rpm), and the combined EtOAc extracts (100 L) were concentrated under reduced pressure to give a crude extract (2027.17 g) for further separation.

Cytotoxicity Assays
MCF-7/ADM, K562/ADM and A549/ADM cells were purchased from Shanghai MEIXUAN Biological Science and Technology Co., Ltd. (Shanghai, China). Cytotoxicity assays were performed via the CCK-8 method in 96-well microplates [30]. A total of 100 µL cells were cultured at 37 • C under a humidified atmosphere of 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal bovine serum and dispersed in replicate 96-well plates with 3 × 10 4 cells/well for 24 h. After incubation, MCF-7/ADM cells were treated with 6.25, 12.5, 25, 50, 100, and 200 µg/mL tested compounds (1)(2) or ADM (used as positive control) for 48 h at 37 • C in a humidified atmosphere of 5% CO 2 , while A549/ADM and K562/ADM cells were treated with six concentration gradients tested compounds (1)(2) or ADM (diluted twice at an initial concentration of 25 µg/mL) for 48 h at 37 • C in a humidified atmosphere of 5% CO 2 . At the end of incubation, each well was added to 10 CCK-8 solution and continued to incubate for 3 h at 37 • C in a humidified atmosphere of 5% CO 2 . Finally, OD was measured at wavelength 490 nm. A total of 10 µL DMSO and 90 µL medium suspension were used as negative control and each assay was performed in triplicate.

Western Blot (WB) Assay
MCF-7/ADM cells were seeded at a density of 2 × 10 5 cells/well in 6-well plates and incubated for 24 h. Each tumor cell was exposed to each test compound at 0, 5, 10, and 20 µg/mL combined with ADM at the concentration of 65 µg/mL in triplicate for 48 h. Cells were washed three times with PBS and lysed directly in the RIPA (Radio Immunoprecipitation Assay) buffer. The lysates were centrifuged at 12,000 rpm for 5 min, and the supernatants were collected. The protein concentrations of the supernatants were determined using a BCA protein assay kit. SDS-PAGE was used to separate whole cell lysates having equivalent amounts of protein to be transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were incubated with primary antibodies and then with HRP-conjugated secondary antibodies. The proteins were then detected using the ECL detection kit.

Data Analysis
Each experiment was performed at least in triplicate. The IC 50 values were calculated by Modified Kou's method. The grayscale values were calculated by IPP 6.0. One-way analysis of variance (one-way ANOVA) was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, and p < 0.0001. The results are expressed as the mean standard deviation (SD) and activity-related figures were calculated by GraphPad Prism 8.0.
Furthermore, sixteen known compounds were also isolated. According to the comparison with the reported NMR data and mass data, sixteen compounds [ Figure 1] were iden-

Cytotoxicity Evaluation In Vitro
The new compounds 1 (PF70) and 2 (PF52) were subjected to cytotoxicity evaluation on the three human cancer cell lines viz MCF-7/ADM, K562/ADM and A549/ADM using the CCK-8 method. Both compounds 1 and 2 showed cytotoxic activity on the cell lines with an inhibition ratio of less than 20% at a concentration lower than 50 µg/mL. The results indicated that both compounds were applicable for studying MDR reversal activity.

MDR Reversal Effects of Compound 1 and 2 In Vitro
The MDR reversal assay was performed using the anticancer drug adriamycin (ADM) at different concentrations combined with the test compounds at concentrations of 0, 5, 10 and 20 µg/mL, respectively, and Vrp as the positive control. The reversal activities of compound 1 and compound 2 against the three tumor cells were shown in Figure 5 and Table 2. In the assay on MCF-7/ADM, both compounds 1 (See Table 2 and Figure 5A) and 2 (See Table 2 and Figure 5B) showed certain reversal effects, but not as effective as those of Vrp. While in the assay on K562/ADM, both compounds 1 (See Table 2 and Figure 5C) and 2 (See Table 2 and Figure 5D) were proved to have significant MDR reversal effects with IC 50 values range from 3.813 ± 0.123 to 2.360 ± 0.208 µg/mL and 3.764 ± 0.297 to 2.448 ± 0.225 µg/mL, respectively, which were higher than that of Vrp with an IC 50 value range from 4.038 ± 0.514 to 2.554 ± 0197 µg/mL at 5, 10, 20 µg/mL, respectively. However, one-way variance contrast analysis (ANOVA) showed no statistical difference (p > 0.05). In the assay on A549/ADM, compound 1 (See Table 2 and Figure 5E) displayed significant MDR reversal effects with IC 50 value range from 2.897 ± 0.072 to 1.841 ± 0.079 µg/mL, which were higher than those of Vrp at 5 and 20 µg/mL, respectively, with IC 50 value ranges from 2.927 ± 0.226 to 1.848 ± 0.015 µg/mL. While the results of the ANOVA indicated no statistical difference (p > 0.05). Compound 2 (See Table 2 and Figure 5F) also showed certain reversal effects, but not as effective as those of Vrp. Meanwhile, as also shown in Figures 6 and 7, the inhibition ratio curves of compound 1 and 2 combined with ADM on the three drug-resistant cell lines indicated that both compounds significantly increased the ADM sensitivities of ADM-resistant cells with significant dosedependence.   the MCF-7/ADM cell line via hindering the overexpression of P-gp. The Western blot assays also showed that the test of compound 2 at the concentrations of 10 and 20 μg/mL, respectively, had remarkable suppression effects on the expression level of P-gp ( Figure  7D) and therefore reversed the MDR of the MCF-7/ADM cell line.    Finally, we tested the P-gp expressions in MCF-7/ADM cells with the treatment of ADM at the concentration of 65 µg/mL combined with compound 1 and 2 at 0, 5, 10, 20 µg/mL, respectively, for 48 h. Our Western blot assays showed that the test with compound 1 at the concentration of 20 µg/mL had a significant suppression effect on the expression level of P-gp ( Figure 6D), which proved that compound 1 reversed the MDR of the MCF-7/ADM cell line via hindering the overexpression of P-gp. The Western blot assays also showed that the test of compound 2 at the concentrations of 10 and 20 µg/mL, respectively, had remarkable suppression effects on the expression level of P-gp ( Figure 7D) and therefore reversed the MDR of the MCF-7/ADM cell line. Figure 5. IC50 values of ADM at different concentrations combined with compound 1 (PF70) and 2 (PF52) at concentrations of 0, 5, 10, 20 μg/mL, respectively: (A) compound 1 (PF70) against MCF-7/ADM; (B) compound 2 (PF52) against MCF-7/ADM; (C) compound 1 (PF70) against K562/ADM; (D) compound 2 (PF52) against K562/ADM; (E) compound 1 (PF70) against A549/ADM; (F) compound 2 (PF52) against A549/ADM. All experiments were performed in triplicate. ns p﹥0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001, and **** p < 0.0001.

Discussion
The strain Tubeufia rubra (PF02-2) was first isolated and identified by Dr. Lu of our research group, so there is no relevant report on the metabolites of the strain. In the paper, two new compounds, viz Rubracin D (1) and E (2), as well as sixteen known constituents (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) were isolated from the crude ethyl acetate extract of the fermentation broth of the fungal strain Tubeufia rubra (PF02-2). Their chemical structures were identified on the basis of their spectroscopic spectra data, as well as via comparison with literature data. Compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) were isolated from the fungal genus Tubeufia for the first time. Bioassay results indicated that the new compounds (1, 2) showed significant MDR reversal activities on MCF-7/ADM, K562/ADM and A549/ADM cell lines. It is worth noting that both compounds (1, 2) were proved to have significant MDR reversal effects mostly higher than those of the positive control viz Vrp in the assay on the K562/ADM cell line. Additionally, compound 1 displayed significant MDR reversal effects mostly higher than those of Vrp in the assay on A549/ADM cell line. The results of the Western blot assays proved that both compounds 1 and 2 significantly reversed the MDR of the MCF-7/ADM cell line via hindering the overexpression of P-gp. We, therefore, speculate that the MDR reversal mechanism of both compounds on the other two resistant cancer cell lines viz K562/ADM
Compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) were isolated from the fungal genus Tubeufia for the first time. Bioassay results indicated that the new compounds (1, 2) showed significant MDR reversal activities on MCF-7/ADM, K562/ADM and A549/ADM cell lines. It is worth noting that both compounds (1, 2) were proved to have significant MDR reversal effects mostly higher than those of the positive control viz Vrp in the assay on the K562/ADM cell line. Additionally, compound 1 displayed significant MDR reversal effects mostly higher than those of Vrp in the assay on A549/ADM cell line. The results of the Western blot assays proved that both compounds 1 and 2 significantly reversed the MDR of the MCF-7/ADM cell line via hindering the overexpression of P-gp. We, therefore, speculate that the MDR reversal mechanism of both compounds on the other two resistant cancer cell lines viz K562/ADM and A549/ADM was also due to inhibiting the overexpression of P-gp.