Rare Chromone Derivatives from the Marine-Derived Penicillium citrinum with Anti-Cancer and Anti-Inflammatory Activities

Three new and rare chromone derivatives, epiremisporine C (1), epiremisporine D (2), and epiremisporine E (3), were isolated from marine-derived Penicillium citrinum, together with four known compounds, epiremisporine B (4), penicitrinone A (5), 8-hydroxy-1-methoxycarbonyl-6-methylxanthone (6), and isoconiochaetone C (7). Among the isolated compounds, compounds 2–5 significantly decreased fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 6.39 ± 0.40, 8.28 ± 0.29, 3.62 ± 0.61, and 2.67 ± 0.10 μM, respectively. Compounds 3 and 4 exhibited cytotoxic activities with IC50 values of 43.82 ± 6.33 and 32.29 ± 4.83 μM, respectively, against non-small lung cancer cell (A549), and Western blot assay confirmed that compounds 3 and 4 markedly induced apoptosis of A549 cells, through Bcl-2, Bax, and caspase 3 signaling cascades.

Human neutrophils are known to play a critical role in the pathogenesis of various inflammatory diseases [19,20]. In response to different stimuli, activated neutrophils secrete a series of cytotoxins, such as superoxide anion (O 2 •-), granule proteases, and bioactive lipids [19,21,22]. Suppression of inappropriate activation of neutrophils by drugs was proposed as a way to combat inflammatory diseases [23].
According to statistics from Taiwan's Ministry of Health and Welfare, cancer remained the top killer in Taiwan for many years [24]. The apoptosis-related proteins, such as Bcl-2, Bax, and caspase-3, regulate cancer cell apoptosis or survival, which was confirmed to be related to many cancers and diseases [25][26][27]. In a preliminary screening, the methanolic extract of P. citrinum showed anti-inflammatory and anti-cancer activities in vitro. The current chemical investigation of this fungus led to the isolation of three new chromone derivatives, epiremisporine C (1), epiremisporine D (2), and epiremisporine E (3), along with four known compounds. The structural elucidation of 1-3 and anti-inflammatory and anti-cancer properties of 1-7 are described herein.

Fermentation, Extraction, and Isolation
In this study, the marine-derived fungal strain Penicillium citrinum (BCRC 09F0458) was cultured in solid-state culturing conditions, in order to enrich the diversity of the fungal secondary metabolites. Chromatographic isolation and purification of the n-BuOH-soluble fraction of an EtOH extract of Penicillium citrinum on a silica gel column and preparative thin-layer chromatography (TLC) obtained three new (1)(2)(3) and four known compounds (4-7) ( Figure 1).

Structure Identification of the Known Isolated Compounds
The known isolated compounds were readily identified by a comparison of physical and spectroscopic data (UV, IR, 1

Biological Studies 2.4.1. Inhibitory Activities on Neutrophil Pro-Inflammatory Responses
The anti-inflammatory effects of the isolated compounds from Penicillium citrinum were evaluated by their ability to suppress formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced O 2 •generation by human neutrophils. The anti-inflammatory activity data are shown in Table 2. The clinically used anti-inflammatory agent, ibuprofen, was used as the positive control. From the results of our anti-inflammatory tests, epiremisporine D (2), epiremisporine E (3), epiremisporine B (4), and penicitrinone A (5) exhibited inhibition (IC 50 values ≤ 8.28 µM) of superoxide anion generation by human neutrophils, in response to fMLP. Thus, our study suggests Penicillium citrinum and its isolated compounds (especially 2, 3, 4, and 5) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases. Table 2. Inhibitory effects of compounds 1-7 from Penicillium citrinum on superoxide anion generation by human neutrophils, in response to fMLP.

New Compound 3 Inhibited Proliferation of A549 Cells
The known compounds, epiremisporine B (4) [16] and penicitrinone A (5) [28], were reported to exhibit anticancer activities in previous studies. Epiremisporine E (3) was selectively tested for clonogenic assay as it is a new compound and possesses cytotoxic activity against A549. The effect of compound 3 on colony formation of A549 cells was examined using the clonogenic assay ( Figure 8). The A549 cell colonies were visualized as blue discs, through crystal violet staining. It was clearly observed that compound 3 (25 µM) significantly reduced the colony formation of A549 cells. Moreover, compound 3 almost completely inhibited the colony formation at 50 µM.

Effects of Epiremisporine E (3) and Epiremisporine B (4) on Protein Expressions of Pro-caspase 3 and Cleaved-caspase 3 in A549 Cells
Caspase 3 activation is a hallmark of apoptosis. Caspase 3 activation involves the cleavage of pro-caspase 3 (the inactive precursor form of caspase 3), leading to the formation of cleaved-caspase 3 (which is the active caspase 3). Upon apoptosis, the pro-caspase 3 would decrease and the cleaved-caspase 3 would increase accordingly. We further investigated whether epiremisporine E (3) and epiremisporine B (4) were able to influence these enzymatic activities of caspase 3. The results showed that compounds 3 and 4 suppressed pro-caspase 3 and increased the cleaved-caspase 3 (Figures 9 and 10). Furthermore, compounds 3 and 4 markedly induced apoptosis of A549 cells through caspase 3-dependent pathways. Figure 9. Western blot analysis for Bcl-2 (a), Bax (b), pro-caspase 3 (c), and cleaved-caspase 3 (d) in each group. Treatment with epiremisporine C (3) significantly reduced the expression levels of Bcl-2 and pro-caspase 3, and increased the expression levels of Bax and cleaved-caspase 3. As-terisks indicate significant differences (* p < 0.05, ** p < 0.01, and *** p < 0.001) compared with the control group.

Effects of Compounds 3 and 4 on Protein Expressions of Bax and Bcl-2 in A549 Cells
To determine whether compounds 3 and 4 could influence the expression of proteins related to A549 cells apoptosis, compounds 3 and 4 (6.25, 12.5, and 25 µM) were added to A549 cells. Figures 9 and 10 showed that the expression level of pro-apoptotic protein, bax was obviously higher with 25 µM treatment of compound 3 or 4 than with 12.5 or 6.25 µM treatment. On the contrary, the cells treated with 25 µM of compound 3 or 4 showed higher Bcl-2 (anti-apoptotic protein) expression than that treated with 12.5 or 6.25 µM. The results showed that compounds 3 and 4 suppressed the expression of Bcl-2 and increased bax expression. Figure 10. Western blot analysis for Bcl-2 (a), Bax (b), pro-caspase 3 (c), and cleaved-caspase 3 (d) in each group. Treatment with epiremisporine B (4) significantly reduced the expression levels of Bcl-2 and pro-caspase 3, and increased the expression levels of Bax and cleaved-caspase 3. As-terisks indicate significant differences (* p < 0.05 and ** p < 0.01) compared with the control group.

Fungal Material
The fungal strain Penicillium citrinum BCRC 09F458 was isolated from waste water, which was collected from Hazailiao, Dongshi, Chiayi, Taiwan, in 2009. The fungal strain was identified as Penicillium citrinum (family Trichocomaceae) by the BCRC center, based on cultural and anamorphic data. The rDNA-ITS (internal transcribed spacer) region was used for further identification. After searching the GenBank database through BLAST (nucleotide sequence comparison program), it was found to have a 100% similarity to P. citrinum. P. citrinum BCRC 09F458 was stored in the Biological Resources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI).

Cultivation and Preparation of the Fungal Strain
We kept P. citrinum BCRC 09F0458 on potato dextrose agar (PDA), and cultivated the strain on PDA at 25 • C for one week, and finally harvested the spores with sterile water. The spores (5 × 10 5 ) were seeded into 300 mL shake flasks containing 50 mL RGY medium (3% rice starch, 7% glycerol, 1.1% polypeptone, 3% soybean powder, 0.1% MgSO 4 , and 0.2% NaNO 3 ), and cultivated with shaking (150 rpm) at 25 • C, for 3 days. After the mycelium enrichment step, an inoculum mixing 100 mL mycelium broth and 100 mL RGY medium was inoculated into plastic boxes (25 cm × 30 cm) containing 1.5 kg sterile rice and cultivated at 25 • C for producing rice, and the above mentioned RGY medium was added for maintaining the growth. After 21 days of cultivation, the rice was harvested, and used as a sample for further extraction.

Biological Assay
The anti-inflammatory effects of the isolated compounds from Penicillium citrinum were evaluated by suppressing fMLP-induced O 2 •generation by human neutrophils. In addition, anti-cancer activity was evaluated by cytotoxicity assay and Western blot analysis.

Preparation of Human Neutrophils
Human neutrophils from the venous blood [21] of healthy, adult volunteers (20-35 years old) were isolated using a standard method of dextran sedimentation, prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes, as previously described [29]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method, were resuspended in HBSS buffer at pH 7.4 and were maintained at 4 • C, prior to use [30].

Measurement of O 2
•-Generation The assay for measurement of O 2 •generation was based on the SOD-inhibitable reduction of ferricytochrome c [31]. In brief, neutrophils (1 × 10 6 cells/mL) pretreated with the various test agents at 37 • C for 5 min were stimulated with fMLP (1 µmol/L) in the presence of ferricytochrome c (0.5 mg/mL). Extracellular O 2 •production was assessed with a UV spectrophotometer at 550 nm (Hitachi U-3010, Tokyo, Japan). The percentage of superoxide inhibition of the test compound was calculated as the percentage of inhibition = {(control − resting) − (compound − resting)}/(control − resting) × 100. The software SigmaPlot was used for determining the IC 50 values [30].

Cells and Culture Medium
A549 (human lung carcinoma) and HT-29 (human colon carcinoma) cells were kindly provided by Prof. T. M. Hu and Prof. Y. Su, respectively, of National Yang-Ming University, Taipei, Taiwan.
All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 µM L-glutamine, and 1 mM sodium pyruvate. The cells were incubated in an atmosphere of 37 • C and 5% CO 2 and passaged twice a week. Cells were stored in liquid nitrogen at −155 • C. After the cells were thawed, the experiment was completed before 30 generations. The purpose was to minimize experimental errors. The compound stock solution was stored in DMSO at a concentration of 10 mM and stored at −20 • C, and finally melted immediately before use.

Cytotoxicity Assay
A MTT colorimetric assay was used to determine cell viability. The assay was modified from that of Mosmann [32]. MTT reagent (0.5 mg/mL) was added onto the attached cells mentioned above (100 µL per 100 µL culture) and incubated at 37 • C for 3 h. Then, DMSO was added and the amount of colored formazan metabolite formed was measured by absorbance at 570 nm, using an ELISA plate reader (µ Quant). The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

Clonogenic Assay
The clonogenic assay followed as previously described with slight changes [33]. For the clonogenic assay, cells at a density of 3000 cells/well were seeded in 6-well plates for 24 h. Next, the cells were treated with compound 3 or vehicle (DMSO) and allowed to form colonies for 14 days. Colonies were washed with PBS, and the cells attached to the plastic surface were fixed in 99% MeOH for 30 min and stained with 0.2% crystal violet for 15 min. The stained cells were quantified using the ImageJ software (BioTechniques, NY, USA).

Statistical Analysis
All data are expressed as mean ± SEM. Statistical analysis was carried out using Student's t-test. A probability of 0.05 or less was considered to be statistically significant. Microsoft Excel 2019 was used for the statistical and graphical evaluation. All experiments were performed at least 3 times.

Data Availability Statement:
The data presented in this study are available in the main text and the supplementary materials of this article.