Chemical Constituents of the Deep-Sea-Derived Penicillium citreonigrum MCCC 3A00169 and Their Antiproliferative Effects

Six new citreoviridins (citreoviridins J–O, 1–6) and twenty-two known compounds (7–28) were isolated from the deep-sea-derived Penicillium citreonigrum MCCC 3A00169. The structures of the new compounds were determined by spectroscopic methods, including the HRESIMS, NMR, ECD calculations, and dimolybdenum tetraacetate-induced CD (ICD) experiments. Citreoviridins J−O (1–6) are diastereomers of 6,7-epoxycitreoviridin with different chiral centers at C-2–C-7. Pyrenocine A (7), terrein (14), and citreoviridin (20) significantly induced apoptosis for HeLa cells with IC50 values of 5.4 μM, 11.3 μM, and 0.7 μM, respectively. To be specific, pyrenocine A could induce S phase arrest, while terrein and citreoviridin could obviously induce G0-G1 phase arrest. Citreoviridin could inhibit mTOR activity in HeLa cells.

Therefore, similar to 1, NOESY, ECD, and ICD experiments and coupling constants analysis were also employed to determine the absolute configurations of 2-6. The geometry at ∆ 8,10,12 double bonds in 2-6 was assigned as E based on the large coupling constants (ca 15 Hz). Likewise, the trans-configuration of epoxides and the cis-configuration for H-6 and H 3 -21 in 2-6 were assigned on the basis of the NOESY correlations of H-6 to H 3 -21 ( Figure 3). The relative configurations of the 2,3,5-trimethyl-tetrahydro-furan-3,4-diol residues in 2-6 were also elucidated based on NOESY correlations, as shown in Figure 3. The ECD spectra of 2 and 5 showed positive Cotton effects at 215 nm and 373 nm and negative Cotton effects at 270 nm (Figure 4), which were in accordance with that of 1. Therefore, 6S,7S-configuration was assigned for 2 and 5. On the contrary, 6R,7Rconfiguration was assigned for 3, 4, and 6 because of the mirror-like ECD spectra (Figure 4). Similar to that of 1, the Mo 2 (OAc) 4  All isolates were tested for antiproliferative activity against HeLa tumor cells. Compounds 7, 14, and 20 exhibited significant effects, with IC 50 values of 5.4 µM, 11.3 µM, and 0.7 µM, respectively ( Figure 6A). To further detect the apoptosis activity of these three compounds, Hela cells were analyzed by western blotting after treatment with 7, 14, and 20 for 40 h. The cleavage of PARP protein, a sensitive apoptotic marker, was used to detect the apoptosis activity. As shown in Figure 6B, they all induced potent apoptosis. It was reported that 20 could inhibit human umbilical vein endothelial cells (HUVECs) proliferation [37]. Compound 7 showed cytotoxicity against several cancer cells, with IC 50 values ranging from 2.6 to 12.9 µM. Terrein (14) displayed strong cytotoxicities against human breast cancer MCF-7 cells [38] and human lung cancer A 549 cells [39]. Hence, our findings were consistent with those reported in previous experiments, though different cancer cell lines were evaluated. To detect their effect on cell cycle progression, HeLa cells were treated with compounds 7, 14, and 20 for 16 h, stained with propidium iodide, and analyzed by flow cytometry. As shown in Figure 7, 7 could induce S phase arrest, while 14 and 20 could obviously induce G0-G1 phase arrest. Compound 20 also inhibited the proliferation of HU-VECs that were arrested at the G0/G1 phase [37]. To detect their effect on cell cycle progression, HeLa cells were treated with compounds 7, 14, and 20 for 16 h, stained with propidium iodide, and analyzed by flow cytometry. As shown in Figure 7, 7 could induce S phase arrest, while 14 and 20 could obviously induce G0-G1 phase arrest. Compound 20 also inhibited the proliferation of HUVECs that were arrested at the G0/G1 phase [37].
(C) To detect their effect on cell cycle progression, HeLa cells were treated with compounds 7, 14, and 20 for 16 h, stained with propidium iodide, and analyzed by flow cytometry. As shown in Figure 7, 7 could induce S phase arrest, while 14 and 20 could obviously induce G0-G1 phase arrest. Compound 20 also inhibited the proliferation of HU-VECs that were arrested at the G0/G1 phase [37]. As is known to all, the mTOR is one of the most usually activated signaling pathways in cancer. The major downstream target of the mTOR is the ribosomal protein S6.
Previously, citreoviridin induces myocardial apoptosis through the PPAR-γ-mTORC2-mediated autophagic pathway [40]. Therefore, compound 20 detected the As is known to all, the mTOR is one of the most usually activated signaling pathways in cancer. The major downstream target of the mTOR is the ribosomal protein S6.
Previously, citreoviridin induces myocardial apoptosis through the PPAR-γ-mTORC2mediated autophagic pathway [40]. Therefore, compound 20 detected the protein level of the phosphorylation of S6. As is shown in Figure 8, compound 20 was found to obviously inhibit p-S6, indicating that 20 could strongly inhibit the mTOR pathway. Therefore, compound 20 might induce apoptosis through mTOR inhibition.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 8 of 12 protein level of the phosphorylation of S6. As is shown in Figure 8, compound 20 was found to obviously inhibit p-S6, indicating that 20 could strongly inhibit the mTOR pathway. Therefore, compound 20 might induce apoptosis through mTOR inhibition.

General Experimental Procedures
Optical rotations were recorded on an Anton Paar MCP 100 polarimeter. ECD spectra were recorded on a Chirascan spectropolarimeter. The HRESIMS spectra were recorded on Q-Exactive Focus tandem mass spectrometry. The NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer. The preparative and semipreparative HPLC were performed on an Agilent Technologies 1260 infinity instrument using ODS or Chiralpak IC columns. UV spectra were recorded on a UV-8000 UV/Vis spectrometer. Column chromatography (CC) was performed on silica gel and Sephadex LH-20. The TLC plates were visualized under UV light or by spraying with 10% H2SO4.

Biological Material
The fungal strain Penicillium citreonigrum was isolated from the deep-sea sediment of the Northeastern Pacific at a depth of −2530 m. The voucher strain was preserved at the Marine Culture Collection of China (MCCC, Xiamen, China) and was given the accession number MCCC 3A00169.

Fermentation and Extraction
P. citreonigrum MCCC 3A00169 was grown under static conditions at 25 °C in 85 × 1

General Experimental Procedures
Optical rotations were recorded on an Anton Paar MCP 100 polarimeter. ECD spectra were recorded on a Chirascan spectropolarimeter. The HRESIMS spectra were recorded on Q-Exactive Focus tandem mass spectrometry. The NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer. The preparative and semipreparative HPLC were performed on an Agilent Technologies 1260 infinity instrument using ODS or Chiralpak IC columns. UV spectra were recorded on a UV-8000 UV/Vis spectrometer. Column chromatography (CC) was performed on silica gel and Sephadex LH-20. The TLC plates were visualized under UV light or by spraying with 10% H 2 SO 4 .

Biological Material
The fungal strain Penicillium citreonigrum was isolated from the deep-sea sediment of the Northeastern Pacific at a depth of −2530 m. The voucher strain was preserved at the Marine Culture Collection of China (MCCC, Xiamen, China) and was given the accession number MCCC 3A00169.

Fermentation and Extraction
P. citreonigrum MCCC 3A00169 was grown under static conditions at 25 • C in 85 × 1 L Erlenmeyer flasks, each containing 80 g of rice and 120 mL of distilled H 2 O. After 47 days, the fermentation broth was extracted by EtOAc three times to give a crude extract (32 g).

ECD Calculation
The conformational analysis was first performed via random searching in the Stochastic using the MMFF94 force field with an energy cutoff of 7.0 kcal/mol and an RMSD threshold of 0.2 Å. All conformers were consecutively optimized at the PM6 and HF/6-31G(d) levels. Dominative conformers were re-optimized at the B3LYP/6-31G(d) level in the gas phase. The theoretical ECD spectra were calculated with the B3LYP method at the 6-311G(d,p) level in MeOH using Gaussian 09. The ECD spectrum was simulated by overlapping Gaussian functions for each transition [41].

Measurement of ICD Spectra
Compounds 1-6 were first dissolved in appropriate DMSO. Then, a quantity of Mo 2 (OAc) 4 were added, with a ligand-to-metal ratio of approximately 1:1.2. The first CD spectrum (CD 0 ) was recorded immediately after mixing and scanned every 10 min until a stationary CD spectrum (CD 1 ) was measured. The induced CD (ICD) spectra were calculated from the CD of the ligand-metal complex (CD 1 ) deducting the inherent CD (CD 0 ).

The Antiproliferative Bioassay
As reported previously, the experiment was conduct using the Cell Counting Kit-8 (CCK-8) assay [42]. Briefly, HeLa cells were seeded in a 96-well plate at a density of 2000 cells/well and were cultured in MEM/EBSS (MEM) containing 10% FBS at 37 • C. After 24 h, the cells were treated with the test compounds, and incubation continued for 72 h. Then, 10 µL CCK-8 solution was added to each well. After incubation at 37 • C for 4 h, the absorbance value of each well was determined using a multi-well plate reader at 450 nm.

Flow Cytometry
After the indicated time treatment, cancer cell arrest was assessed by an FACScan flow cytometer (Beckman Coulter, California, USA), following the manual procedure. The cells were harvested by trypsin digestion, washed with PBS, and fixed with ice-cold 70% ethanol at 4 • C overnight. The fixed cells were then washed twice in PBS and treated for 30 min at RT with propidium iodide in PBS and analyzed. Flow cytometry data were analyzed using CytExpert (Beckman Coulter).

Western Blot Analysis
For Western blot assays, HeLa cells were treated with the compounds for the indicated time. Then, the cells were harvested, lysed, and centrifuged at 12,000 rpm/min for 10 min. The supernatant was added with a 1/5 volume of 5 × SDS and boiled. After electrophoresis, protein samples were transferred to the PVDF film, blocked with fat-free milk, incubated with the first antibody and washed, and then incubated with the secondary antibody and washed. Then, the ECL droplets were reacted on the membrane surface, and the bands were imaged by the multifunctional chemiluminescence imaging system.