Constituents and Anti-Multidrug Resistance Activity of Taiwanofungus camphoratus on Human Cervical Cancer Cells

Resistance to anti-cancer drugs is one of the main factors of treatment failure resulting in high morbidity. Among the reasons of resistance, overexpression of efflux pumps leading to multidrug resistance is an important issue that needs to be solved. Taiwanofungus camphoratus has been used as a nutritional supplement to treat various cancers. However, its effects on the resistance to chemotherapeutic agents are still unknown. In this study, we report four new chemical constituents of T. camphoratus isolated from an ether extract: camphoratins K (1) and N (2) and benzocamphorins G (3) and I (4). Furthermore, we evaluated zhankuic acids A–C for their P-glycoprotein (P-gp) inhibitory effects. The results showed that zhankuic acid A was the most potent P-gp inhibitor compound and (at 20 μM) could reverse drug resistance in human cancer cells, restoring an IC50 of 78.5 nM for doxorubicin, of 48.5 nM for paclitaxel, and of 321.5 nM for vincristine, indicating a reversal fold of 48, 38, and 45 times, respectively. This study provides support for the use of T. camphoratus in the further development of cancer therapy.


Introduction
Treatment failure or metastasis are still the leading causes of death for cancer patients [1]. One of the important factors causing treatment failure is drug resistance, which can be intrinsic or acquired [2,3]. Cancer stem cells [4], tumor microenvironment, and host effects are the main reasons of intrinsic resistance, while efflux pumps, alteration of drug targets, degradation of anticancer drugs, and DNA self-repair cause acquired resistance. In particular, overexpression of efflux pumps leads to multidrug resistance (MDR) [2,3]. In order to increase the efficacy of anticancer drugs, strategies to reverse multidrug resistance are extensively studied, and P-glycoprotein (P-gp) inhibitors have evolved to the fourth generation [5][6][7][8]. However, because of their inability to of improve drug efficacy in patients as well as their toxicity, P-gp inhibitors are not yet available in the clinic. Therefore, research is now focusing on natural products, hoping to find safe and effective P-gp inhibitors [5][6][7][8].
The major chemical constituents of this fungus are triterpenoids [17][18][19] and benzenoids [20,21]. Other components are steroids [22], diterpenoids [23], terpenoids [24], lignans [22], maleic and succinic acid derivatives [25], etc. Although the extract of T. camphoratus has been used as a nutritional supplement for treating cancers, the P-gp inhibitory effects of its main constituents are still unknown. Therefore, in addition to reporting new chemical constituents of T. camphoratus, this study reveals the P-gp inhibitory effects of zhankuic acids A-C.

P-gp Inhibitory Effects of the Extract of T. camphoratus
A pilot study using methanol as a solvent was done to evaluate the P-gp inhibitory effects of the extract of T. camphoratus ( Figure 5). The methanol extract was further partitioned using water and EtOAc. Therefore, the methanol extract (TAM), the EtOAc layer (TAE), and the water layer (TAW) were tested using human stably P-gp-expressing cells (ABCB1/Flp-InTM-293) in a calcein AM (acetoxymethyl) uptake assay [26]. The increased intracellular calcein fluorescence corresponded to the inhibition level of P-gp efflux function. The methanol extract as well as the EtOAc layer and the water layer exhibited P-gp inhibitory activities at concentrations of 10 and 20 μM. The methanol extract and the EtOAc layer exhibited inhibition in a dose-dependent manner. Moreover, the methanol extract at 20 μM (TAM 20) showed P-gp inhibition comparable to that of the first-generation P-gp inhibitor verapamil at a concentration of 2.5 μM.

Zhankuic Acids A-C Inhibited P-gp Efflux Function
Although four new compounds were isolated, they were in little amount. In order to better understand the main P-gp inhibitory effects of T. camphoratus, three of its major components, zhankuic acids (ZAs) A-C, were evaluated for their ability to inhibit P-gp using the calcein AM uptake assay. ZAs A, B, C inhibited P-gp efflux function in a concentration-dependent manner ( Figure 6). Among the tested compounds, ZA-A demonstrated the most significant P-gp inhibitory effect.

P-gp Inhibitory Effects of the Extract of T. camphoratus
A pilot study using methanol as a solvent was done to evaluate the P-gp inhibitory effects of the extract of T. camphoratus ( Figure 5). The methanol extract was further partitioned using water and EtOAc. Therefore, the methanol extract (TAM), the EtOAc layer (TAE), and the water layer (TAW) were tested using human stably P-gp-expressing cells (ABCB1/Flp-InTM-293) in a calcein AM (acetoxymethyl) uptake assay [26]. The increased intracellular calcein fluorescence corresponded to the inhibition level of P-gp efflux function. The methanol extract as well as the EtOAc layer and the water layer exhibited P-gp inhibitory activities at concentrations of 10 and 20 µM. The methanol extract and the EtOAc layer exhibited inhibition in a dose-dependent manner. Moreover, the methanol extract at 20 µM (TAM 20) showed P-gp inhibition comparable to that of the first-generation P-gp inhibitor verapamil at a concentration of 2.5 µM.

P-gp Inhibitory Effects of the Extract of T. camphoratus
A pilot study using methanol as a solvent was done to evaluate the P-gp inhibitory effects of the extract of T. camphoratus ( Figure 5). The methanol extract was further partitioned using water and EtOAc. Therefore, the methanol extract (TAM), the EtOAc layer (TAE), and the water layer (TAW) were tested using human stably P-gp-expressing cells (ABCB1/Flp-InTM-293) in a calcein AM (acetoxymethyl) uptake assay [26]. The increased intracellular calcein fluorescence corresponded to the inhibition level of P-gp efflux function. The methanol extract as well as the EtOAc layer and the water layer exhibited P-gp inhibitory activities at concentrations of 10 and 20 μM. The methanol extract and the EtOAc layer exhibited inhibition in a dose-dependent manner. Moreover, the methanol extract at 20 μM (TAM 20) showed P-gp inhibition comparable to that of the first-generation P-gp inhibitor verapamil at a concentration of 2.5 μM.

Zhankuic Acids A-C Inhibited P-gp Efflux Function
Although four new compounds were isolated, they were in little amount. In order to better understand the main P-gp inhibitory effects of T. camphoratus, three of its major components, zhankuic acids (ZAs) A-C, were evaluated for their ability to inhibit P-gp using the calcein AM uptake assay. ZAs A, B, C inhibited P-gp efflux function in a concentration-dependent manner ( Figure 6). Among the tested compounds, ZA-A demonstrated the most significant P-gp inhibitory effect. (a) (b) (c)

Zhankuic Acids A-C Inhibited P-gp Efflux Function
Although four new compounds were isolated, they were in little amount. In order to better understand the main P-gp inhibitory effects of T. camphoratus, three of its major components, zhankuic acids (ZAs) A-C, were evaluated for their ability to inhibit P-gp using the calcein AM uptake assay. ZAs A, B, C inhibited P-gp efflux function in a concentration-dependent manner ( Figure 6). Among the tested compounds, ZA-A demonstrated the most significant P-gp inhibitory effect.

The MDR Reversal Effects of ZAs A, B, C
To examine the MDR reversal effects of ZAs A, B, C, the cytotoxicity of a combination of these triterpenoids and chemotherapeutic drugs was evaluated in HeLaS3 and MDR KBvin cells. The IC50 of doxorubicin, paclitaxel, and vincristine in HeLa cells were 104 nM, 4.65 nM, and 41.5 nM, while in KBvin cells they were 3750 nM, 1824 nM, and 14,540 nM, indicating high multidrug resistance of the cells. When ZAs A-C were combined with the chemotherapeutic agents, the IC50 of doxorubicin, paclitaxel, and vincristine in MDR KBvin cells were significantly decreased (Table 1). Reversal folds were calculated by dividing the IC50 of the individual chemotherapeutic drug by the IC50 of the compound-drug combinations. ZA-A possessed the most significant MDR reversal effect among the tested compounds. It (20 μM) reversed drug resistance leading to an IC50 of 78.5 nM for doxorubicin, of 48.5 nM for paclitaxel, and of 321.5 nM for vincristine, corresponding to reversal folds of 48, 38, and 45, respectively. 1.900 ± 0.14* 2.4 228.5 ± 2.23* 8 Figure 6. The concentration-dependent inhibitory effects of zhankuic acids (ZAs) A, B, C on P-gp in ABCB1/Flp-In TM -293 cells. * denotes p < 0.05 compared with the intracellular calcein fluorescence in control group.

The MDR Reversal Effects of ZAs A, B, C
To examine the MDR reversal effects of ZAs A, B, C, the cytotoxicity of a combination of these triterpenoids and chemotherapeutic drugs was evaluated in HeLaS3 and MDR KBvin cells. The IC 50 of doxorubicin, paclitaxel, and vincristine in HeLa cells were 104 nM, 4.65 nM, and 41.5 nM, while in KBvin cells they were 3750 nM, 1824 nM, and 14,540 nM, indicating high multidrug resistance of the cells. When ZAs A-C were combined with the chemotherapeutic agents, the IC 50 of doxorubicin, paclitaxel, and vincristine in MDR KBvin cells were significantly decreased (Table 1). Reversal folds were calculated by dividing the IC 50 of the individual chemotherapeutic drug by the IC 50 of the compound-drug combinations. ZA-A possessed the most significant MDR reversal effect among the tested compounds. It (20 µM) reversed drug resistance leading to an IC 50 of 78.5 nM for doxorubicin, of 48.5 nM for paclitaxel, and of 321.5 nM for vincristine, corresponding to reversal folds of 48, 38, and 45, respectively.

General
The spectroscopic data of the purified compounds including optical rotations ([α] 25 D ), UV, and IR spectra were recorded on a Jasco P-2000 digital polarimeter (Jasco, Tokyo, Japan), a Hitachi U-0080D diode array spectrophotometer (Hitachi, Tokyo, Japan), and a Jasco FT/IR-4100 spectrophotometer (Jasco, Tokyo, Japan), respectively. The mass spectra were collected on a Shimadzu LC-MS 8040 spectrometer (Shimadzu, Kyoto, Japan). The HRMS data were obtained on a JMS-T100LP spectrometer (Jeol, Tokyo, Japan). 1 H-, 13 C-, and 2D NMR spectra were recorded on the Bruker AV-500 and Avance III-400 NMR spectrometers (Bruker, Billerica, MA, USA). The deuterated solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals used in this study were provided by Merck KGaA (Darmstadt, Germany). Column chromatography was performed on silica gels in different mesh sizes (70-230 and 230-400 mesh, Kieselgel 60, Merck KGaA, Darmstadt, Germany). Thin-layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck KGaA, Darmstadt, Germany). The spots on TLC were detected by UV light or spraying with 10% (v/v) H 2 SO 4 followed by heating at 110 • C for 10 min.

Culture of Cell Lines
Human stably P-gp-expressing cells (ABCB1/Flp-InTM-293) were established and cultured in DMEM as in a previous study [27]. The human cervical epithelioid carcinoma cell line HeLaS3 was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan), and the multi-drug resistant human cervical cancer cell line KBvin was kindly provided by Dr. Kuo-Hsiung Lee (University of North Carolina, Chapel Hill, NC, USA). All cancer cell lines were cultured in RPMI-1640 containing 10% FBS, at 37 • C in a humidified atmosphere of 5% CO 2 .

Calcein AM Uptake Assay
The calcein AM uptake assay was performed to evaluate the inhibitory effect of the test compounds on human P-gp efflux function. To be brief, 1 × 10 5 cells/well were seeded in 96-well black plates overnight. Before starting the assay, the cells were washed and pre-incubated with warm Hanks balanced salt solution (HBSS) for 30 min. Then, the test compounds were added, and incubation was carried out for 30 min. Calcein-AM was added after washing with warm PBS. The BioTek Synergy HT Multi-Mode Microplate Reader was utilized to detect calcein fluorescence (excitation/emission wavelength = 485 nm/528 nm) at 37 • C every 3 min for 30 min. Each experiment was performed at least three times, each in triplicate on different days.

SRB Cytotoxicity Assay and Reversal Fold Calculation
Briefly, after 72 h of treatment with series concentrations of chemotherapeutic drugs with or without the test compounds, 50% trichloroacetic acid (TCA) was added to fix the cells for 30 min. After air-drying, the cells were stained with 0.04% sulforhodamine B (SRB) for 30 min and washed with 1% acetic acid. The bound stain was solubilized in 10 mM Tris base, and the absorbance was measured by a Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) at 515 nm. Reversal folds were calculated by dividing the IC 50 of each drug by the IC 50 of the compound-drug combination treatment.

Conclusions
Although numerous anti-cancer drugs are marketed, resistance to cancer treatments is still the top reason for cancer death. Multidrug resistance is largely due to the high expression of efflux pumps. T. camphoratus, a medicinal fungus, was reported to exhibit anti-cancer properties, but its effects toward cancer multidrug resistance are unknown. In this study, four new chemical constituents, camphoratins K (1) and N (2) and benzocamphorins G (3) and I (4), were reported for the first time, and the main constituents of T. camphoratus, zhankuic acids A-C, were found to have P-gp inhibitory effects in a dose dependent manner. In addition, zhankuic acid A (20 µM), the most potent P-gp inhibitor, could effectively reverse MDR in KBvin cells, leading to an IC 50 of 78.5 nM for doxorubicin, of 48.5 nM for paclitaxel, and of 321.5 nM for vincristine, corresponding to reversal folds of 48, 38, and 45, respectively.