The fungus P. oxalicum was purified from marine sediments collected from the southeast coastal region of China and identified through internal transcribed spacer analysis by Beijing Sunbiotech Co. Ltd. The specimen was preserved in the China Center for Type Culture Collection (preservation no. CCTCC M2013714). 4,4′-SAD was prepared after the fermentation and extraction of P. oxalicum and successive purification by chromatographic techniques; the detailed procedure was described in the authors' previous study (9). The structure of 4,4′-SAD is shown in Fig. 1A.

PLC/PRF/5 and HuH-7 liver cancer cell lines were purchased from the Shanghai Cell Resource Center. The murine H22 hepatoma cell line was obtained from the Cancer Metastasis Alert and Prevention Center, Fuzhou University. All cell lines were cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences) or DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences) at 37°C in a 5% (v/v) CO₂ humidified atmosphere.

A total of 32 female Kunming (KM) mice (weight, 18–20 g; age, 6 weeks) were purchased from Minhou County Wu Experimental Animal Trade Co., Ltd. The mice were maintained under controlled conditions at 19–23°C and 40–60% humidity, with a 12-h light/dark cycle and free access to drinking water and food. Animal experiments were approved by the Animal Care and Use Committee of the Institute of Biomedical and Pharmaceutical Technology (Fuzhou University), according to the Guide for the Care and Use of Laboratory Animals (15). All mice were euthanized with an overdose of sodium pentobarbital (150 mg/kg; intraperitoneally).

Cells were harvested, washed with PBS and suspended at 1×10⁶ cells/ml in Hank's balanced salt solution supplemented with 3% FBS and 10 mM HEPES. Cells were incubated at 37°C for 90 min with 15 µg/ml Hoechst 33342 (Sigma-Aldrich; Merck KGaA) alone or in the presence of 50 µM verapamil (Sigma-Aldrich; Merck KGaA). During the incubation, the tubes were shaken every 20 min to mix the cells with the solution. The cells were washed twice with Hank's solution, 1 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) was added and this was filtered through a 70 µm cell strainer (BD Biosciences; Becton, Dickinson and Company). SP cell analysis and sorting were performed using MoFlo XDP (Beckman Coulter, Inc.). The data were analyzed using Summit software version 5.0 (Beckman Coulter, Inc.). Hoechst 33342 was excited with a UV laser at 350 nm and fluorescence emission was measured with 450 nm (Hoechst blue) and 570 nm (Hoechst red) optical filters. PI labeling was measured for the discrimination of dead cells.

The water-soluble tetrazolium salt (WST-1; Roche Diagnostics) assay was used to assess cytotoxicity as described previously (9). Briefly, 4×10³ cells per well in a 96-well plate were cultured at 37°C for 24 h and incubated with gradient concentrations of cisplatin (Sigma-Aldrich; Merck KGaA; 0, 2, 4, 8, 16, 32 and 64 µM), doxorubicin (Sigma-Aldrich; Merck KGaA; 0, 0.5, 1, 1.5, 2, 3 and 4 µM) or 4,4′-SAD (0, 0.5, 1, 1.5, 2, 3 and 4 µM) at 37°C for 48 h. Then, cells were treated with 10% WST-1 reagent for 4 h at 37°C and the optical density at 450 nm was tested with an ELISA reader (TECAN Infinite M200 Pro; Tecan Group, Ltd.). Each experiment was carried out in triplicate. The IC₅₀ was calculated using GraphPad Prism software version 7.0 (GraphPad Software, Inc.).

The wound healing assay was performed to assess cell motility as described in our previous study (9). PLC/PRF/5 and HuH-7 cells (8×10⁵ cells/ml) were seeded in 6-well plates and incubated for 24 h to form confluent cell clusters. The vertical cell wounds were generated by 10 µl sterile microtips, washed with PBS three times to remove shedding cells and cultured with 2 ml FBS-free DMEM containing different concentrations (0, 0.5 and 1 µM) of 4,4′-SAD. Wound closure was observed at 0, 24 and 48 h with an inverted fluorescent microscope (magnification, ×100; Nikon Corporation). Each experiment was performed in triplicate.

Inhibition capacity against cell invasion was assessed using the Millipore 24-well Millicell Chamber with an 8-µm pore size (EMD Millipore). Using a standard procedure, the upper of Transwell chambers were coated with Matrigel (BD Biosciences; Becton, Dickinson and Company) at 37°C for 1 h and a 100 µl cell suspension containing a total of 2×10⁵ cells (previously treated with 0, 0.5, 1 and 2 µM 4,4′-SAD) in DMEM without FBS were added to the upper chamber. The lower chamber was filled with 600 µl DMEM with 20% FBS. After incubation at 37°C for 24 h, the chamber was washed twice with PBS buffer, fixed with precooled 100% methanol on ice for 15 min and stained with 0.5% crystal violet at room temperature for 20 min. In total, five random fields were imaged and the number of invaded cells was counted under a fluorescent microscope (magnification, ×400; Nikon Corporation).

Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated with 12% SDS-PAGE by electrophoresis (Bio-Rad Laboratories, Inc.). The protein sample was transferred to the nitrocellulose membrane, which was blocked with 5% (w/v) milk-PBS-0.1%Tween 20 (TBST) at room temperature for 1 h and incubated with specific primary rabbit antibodies against human (h)ABCG2 (cat. no. 42078S; Cell Signaling Technology, Inc.), hMMP-9 (cat. no. 3852S; Cell Signaling Technology, Inc.), hTIMP-1 (cat. no. 8946S; Cell Signaling Technology, USA), mouse (m)MMP-9 (cat. no. BA2202; Wuhan Boster Biological Technology, Ltd.), mTIMP-1 (cat. no. A00561; Wuhan Boster Biological Technology, Ltd.) and β-actin (cat. no. 3700; Cell Signaling Technology, Inc.) at 1:1,000 dilutions in 5% (w/v) milk-PBST overnight at 4°C. The membranes were subsequently washed three times, every 10 min, in PBST and incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5,000; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) secondary antibody or an HRP-conjugated anti-mouse IgG (1:5,000; cat. no. BA1050; Wuhan Boster Biological Technology, Ltd.) secondary antibody at room temperature for 1 h. Protein bands were detected by the FluorChem E digital darkroom system (ProteinSimple). Blots were performed in triplicate and protein expression levels were quantified using ImageJ v.1.8.0 software (National Institutes of Health).

Total RNA was isolated from cells with TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and mRNA was synthesized to cDNA with the GoScript Reverse Transcriptase system (Promega Corporation) at 42°C for 1 h. qPCR was subsequently performed using a Light Cycler 480 II (Roche Diagnostics) with the miScript SYBR Green PCR kit (Qiagen GmbH). The following thermocycling conditions were used: Initial denaturation at 95°C for 2 min; followed by 40 cycles at 94°C for 15 sec, 55°C for 15 sec and 68°C for 30 sec. The oligonucleotide primer pairs used in this study were as follows: ABCG2 forward, 5′-CAGGTGGAGGCAAATCTTCGT-3′ and reverse, 5′-ACCCTGTTAATCCGTTCGTTTT-3′; MMP-9 forward, 5′-CCTGGAGACCTGAGAAC-3′ and reverse, 5′-CAGGGACAGTTGCTTCT-3′; TIMP-1 forward, 5′-ACTCTTGCACATCACTACCT-3′ and reverse, 5′-CATTACTCACAGTCCCTTTT-3′; and GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′. The relative mRNA expression levels were quantified using the 2^−ΔΔCq method (16).

To assess the effects of 4,4′-SAD on tumor metastasis in vivo, the present study established a H22 i.v. model. In total, 4×10⁴ H22 cells in 200 µl PBS were injected into the tail vein of KM mice. KM mice were randomly divided into four groups (n=8 for each group) on day 1. Dry compounds were suspended in 1% carboxymethylcellulose (CMC)-Na (Sigma-Aldrich; Merck KGaA) for immediate use. Mice were orally gavaged with either 200 µl normal 1% CMC-Na solution (control) or 4,4′-SAD (10, 20 or 40 mg/kg) every 2 days from days 2–28. Weight was measured every 4 days until mice were euthanized on day 28. Contrastive images were captured after eviscerating the mice lungs. Lungs were excised, fixed in 10% neutral buffered formalin for 24 h at room temperature, embedded in paraffin, cut into 3–5 µm sections and stained with hematoxylin for 5 min and eosin for 1 min, both at room temperature. The number of tumor nodules on the lung surface was counted by visual inspection using a magnifying glass and tissues were observed under a fluorescent microscope (magnification, ×400; Nikon Corporation).

Tumor tissues of lungs in KM mice were harvested, fixed in 10% neutral buffered formalin at room temperature for 24 h, then embedded in paraffin and cut into 3-µm thick sections. The sections were blocked with 5% (w/v) BSA (EMD Millipore) at room temperature for 30 min and incubated with anti-MMP-9 antibody (1:100; cat. no. BA2202; Wuhan Boster Biological Technology, Ltd.) overnight at 4°C as previously described (17). Following the primary antibody incubation, the sections were incubated with an HRP-conjugated anti-rabbit IgG secondary antibody (1:500; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) at room temperature for 30 min. The expression of MMP-9 was determined with the 3,3-diaminobenzidine assay kit (OriGene Technologies, Inc.), according to the manufacturer's protocol. All sections were imaged using a light microscope (magnification, ×400).

All data are presented as the mean ± SD of triplicate repeats. All statistical analyses were conducted using a Student's t-test or one-way ANOVA followed by Tukey's test, using SPSS 13.0 software (IBM Corp.). P<0.05 was considered to indicate a statically significant difference.

The present study isolated SP and non-SP (NSP) cells from human liver cancer cell lines PLC/PRF/5 and HuH-7 based on the ability of SP cells to actively efflux the Hoechst 33342 dye. To evaluate the cytotoxic effects of 4,4′-SAD and traditional chemotherapy drugs on SP and NSP cells, cells were treated with gradient concentrations of 4,4′-SAD, cisplatin and doxorubicin for 48 h, then cell viability was assessed via the WST-1 assay. It was found that the IC₅₀ values of cisplatin, doxorubicin and 4,4′-SAD were 23.63, 1.731 and 1.871 µM in PLC/PRF/5 SP cells and 18.89, 1.894 and 1.088 µM in HuH-7 SP cells, respectively (Fig. 1B and C). Therefore, suggesting that SP cells were more sensitive to 4,4′-SAD compared with traditional chemotherapeutic drugs. Furthermore, the IC₅₀ values of cisplatin and doxorubicin were higher in SP cells compared with NSP cells in both cell lines, whereas the IC₅₀ value of 4,4′-SAD was lower in SP cells compared with NSP cells (Fig. 1B and C), thus indicating that SP cells were resistant to traditional chemotherapy drugs, but still sensitive to 4,4′-SAD.

The effect of 4,4′-SAD and the traditional chemotherapy drugs, cisplatin and doxorubicin, on the percentage alternation of SP cells was examined in PLC/PRF/5 and HuH-7 cells. It was found that the fraction of SP cells increased from 2.95 to 3.88 and 3.63%, but decreased to 0.95% in PLC/PRF/5 after the 48 h treatment of 10 µM cisplatin, 1 µM doxorubicin and 1 µM 4,4′-SAD, respectively (Fig. 1D). Moreover, the fraction of SP cells increased from 2.16 to 3.15 and 3.98%, but decreased to 1.09% in HuH-7 after 48 h treatment of 10 µM cisplatin, 1 µM doxorubicin and 1 µM 4,4′-SAD, respectively (Fig. 1D). Collectively, the results demonstrated that 4,4′-SAD inhibited and had cytotoxic effects on SP cells, whereas the traditional chemotherapy drugs cisplatin and doxorubicin had more powerful cytotoxic effects on NSP cells compared with SP cells.

Besides drug resistance, SP cells are also involved in tumor invasion and metastasis (18). Due to the anti-SP cell efficacy of 4,4′-SAD in vitro, the present study investigated whether 4,4′-SAD could affect the invasion and migration of PLC/PRF/5 and HuH-7 SP cells using wound healing and Transwell assays. The wound healing assay results demonstrated a decrease in the migration of both PLC/PRF/5 and HuH-7 SP cells after treatment with gradient concentrations of 4,4′-SAD for 24 and 48 h (Fig. 2A). However, 4,4′-SAD was more effective at suppressing of SP cell migration in PLC/PRF/5 cells compared with HuH-7 cells.

In the Transwell assay, the number of PLC/PRF/5 SP cells passing through the Matrigel per field under an inverted microscope (magnification, ×100) was reduced from 203±13 to 141±6, 107±4 and 86±5 after treatment with 0, 0.5, 1 and 2 µM 4,4′-SAD for 24 h, respectively. Moreover, the number of invaded HuH-7 SP cells per field decreased from 147±7 to 57±4, 28±5 and 23±3 after treatment with 0, 0.5, 1 and 2 µM 4,4′-SAD for 24 h, respectively (Fig. 2B). Therefore, it the results indicated that 4,4′-SAD significantly decreased the invasion of PLC/PRF/5 and HuH-7 SP cells.

4,4′-SAD decreases the expression levels of ABCG2 and MMP-9 and increases the expression of TIMP-1 in SP cells. To further examine the role of 4,4′-SAD in inhibiting the proliferation, migration and invasion of SP cells, related gene expression levels were measured by western blotting and RT-qPCR. As ABCG2 is an efflux protein and multidrug resistance marker in SP cells, ABCG2 expression was assessed and it was found that 4,4′-SAD reduced the protein and mRNA expression levels of ABCG2 in SP cells in a concentration-dependent manner (Fig. 3A). The results also indicated that 4,4′-SAD treatment downregulated the expression of MMP-9 at both the protein and mRNA levels, and upregulated the expression of TIMP-1, an inhibitor of MMP-9, in a dose-independent manner (Fig. 3B). Thus, the results suggested that 4,4′-SAD inhibited metastasis by reducing the proteolytic degradation of the extracellular matrix (ECM) and basement membrane near the tumor.

The antimetastatic efficacy of 4,4′-SAD was further assessed using the H22 HCC model in mice. Firstly, the present study investigated whether the role of 4,4′-SAD on metastasis in murine H22 cells was the same as in human PLC/PRF/5 and HuH-7 cells in vitro. It was found that 4,4′-SAD attenuated the expression of MMP-9 and enhanced the expression of TIMP-1 in H22 cells (Fig. S1), which indicated that 4,4′-SAD inhibited the metastasis of mouse H22 cells by regulating MMP-9 and TIMP-1.

The mouse model was constructed by injecting H22 into the tail vein of KM mice at 4×10⁴ cells in 200 µl PBS. Then, mice were orally gavaged with either normal CMC-Na (control) or 4,4′-SAD (10, 20 or 40 mg/kg, every other day) from days 2–28 and were euthanized on day 28 (Fig. 4A). The results suggested that mice treated with 4,4′-SAD exhibited less lung metastatic loci compared with mice in the control group (Fig. 4B and C). Moreover, no significant reduction in body weight change was observed after 4,4′-SAD treatment (Fig. 4D). Histological examinations, which identified the presence of HCC cells in the lung parenchyma, demonstrated the presence of metastasis (Fig. 4E). Furthermore, immunohistochemical staining indicated that MMP-9 expression in lung tissues was inhibited in a dose-independent manner, which was consistent with the experimental results at the cellular level (Fig. 4E). Collectively, these results suggested that 4,4′-SAD efficiently suppressed lung metastasis without obvious side effects in vivo.