Andrastone A From the Deep-Sea-Derived Fungus Penicillium allii-sativi Acts as an Inducer of Caspase and RXRα-Dependent Apoptosis

Two new (1, 2) and one known (3) meroterpenoids were isolated from the deep-sea-derived fungus Penicillium allii-sativi. The relative structures of new compounds were determined on the basis of an extensive analysis of the NMR and MS data, and the absolute configurations were established by ECD calculations. Andrastone A (1) is a rare andrastin bearing an unusual cyclopentan-1,3-dione. It shows a selectively antiproliferative effect against HepG2 tumor cells with an IC50 value of 7.8 μM. Mechanism study showed that apoptosis via Caspase and RXRα pathways are responsible for the inhibitory effect.


INTRODUCTION
The ocean yields an impressive array of novel compounds with exquisite structures and potent bioactivities (Blunt et al., 2017(Blunt et al., , 2018. Interestingly, more and more evidence shows that marine natural compounds are mainly produced by marine microorganisms. In 2017, 57% of the total new marine natural products were reported from marine microorganisms (Carroll et al., 2019). As our research investigating bioactive secondary metabolites from the deep-sea-derived microorganisms continues, a series of interesting compounds were obtained (Yang et al., 2013;Niu et al., 2017;Xie et al., 2019). The crude extract of Penicillium allii-sativi, a fungus isolated from the deep-sea water of the western Pacific, showed significant antiproliferative activities against several cancer cell lines. Therefore, a chemical investigation was conducted, which led to the discovery of two new and one known meroterpenoids (Figure 1), among which compound 1 showed potent effects. Herein, we report the isolation, structure, and bioactivities of these compounds.

General Experimental Procedures
The NMR spectra were recorded on a Bruker 400 MHz spectrometer using TMS as an internal standard. The HRESIMS spectra were measured on a Waters Xevo G2 Q-TOF (Waters) mass spectrometer. Optical rotations were measured with an Anton Paar MCP100 polarimeter. CD spectra were measured on a JASCO J-715 spectropolarimeter. The semipreparative HPLC was carried out on an Agilent technologies 1260 infinity instrument equipped with DAD detector (Agilent, USA) using a reversedphase C18 column (5 µm, 10 × 250 mm; Cosmosil, Japan). Column chromatography (CC) was performed on silica gel, Sephadex LH-20 (Amersham Pharmacia Biotech AB), and ODS (50 µm, Daiso, Japan).

Fermentation, Extraction, and Isolation
Penicillium allii-sativi was isolated from the deep-sea water of the western Pacific at a depth of −4,302 m, in 2012. The voucher strain is preserved at the Marine Culture Collection of China (Xiamen, China) with the accession number of MCCC 3A00580.
The fungus was cultured on a PDA plate at 25 • C for 3 days. The fresh mycelia were then cultured in 50 × 1 L Erlenmeyer flasks under static conditions at 28 • C, each containing 80 g rice and 120 mL distilled water. After 30 days, the fermented broth was extracted with EtOAc for three times. The organic solvent was evaporated under reduced pressure to afford an organic extract (120 g). The extract was partitioned by MeOH and then extracted with petroleum ether (PE). The MeOH layer was concentrated to provide a defatted extract (60.4 g).

X-Ray Crystallographic Analysis of Compound 3
Compound 3 was obtained as a colorless non-merohedral twin crystal. The crystal data were recorded with an Xcalibur Eos Gemini single-crystal diffractometer with Cu Kα radiation (λ = 1.54184 Å). Space group C2 (5)

Apoptosis Determination
After treated with 1 (5 µM) for 24 h, HepG2 cells were stained with 5 µL propidium iodide (PI) by adding 5 µL Annexin V-APC, in darkness for 15 min. The apoptosis results were analyzed using a FAC scan flow cytometer (Becton Dickinson, Sparks, Maryland, USA), as described previously (Xie et al., 2019). Paclitaxel was used as the positive control. H (400 MHz) and 13 C (100 MHz) NMR spectroscopic data of compounds 1-3 (δ in ppm, J in Hz within the parenthesis).

RXRα Transcriptional Activity
As reported previously (Duan et al., 2010), the two target plasmids (30 ng pBind RXRα LBD and 60 ng PG5 LUC) were transfected by Liposome 2000 (Invitrogen) in the cell. The cells were then exposed to 1 (10, 20, 40 µM) for 16 h. According to the introduction of the Dual-Luciferase Reporter Assay System kit (promega), the activities of Firefly luciferase (FL) and Rellina luciferase (RL) were checked. The fold activities were calculated as the relative luciferase activities ratio between sample and blank control.

RESULTS AND DISCUSSION
Compound 1 was obtained as colorless oil. The molecular formula of C 28 H 40 O 7 was determined according to the sodium adduct ion peak at m/z 511.2672 [M + Na] + in its HRESIMS spectrum, indicating nine degrees of unsaturation. The 1 H and 13 C NMR spectra (Supplementary Material) showed 28 carbon signals including eight methyls, one methoxyl, four methylenes, four methines (one olefinic, one oxygenated, and two aliphatic), and 11 non-protonated carbons (two ketones, two carboxyls, one olefinic, one oxygenated, and five aliphatic). Except for the typical signals of one methoxyl group [δ H 3.54 (3H, s); δ C 51.8 q] and one acetoxyl moiety [δ H 2.03 (3H, s); δ C 21.1 q, 170.0 s] group, the remaining signals indicated a 25-carbon skeleton of 1. Since two ketones, two carboxyls, and one olefinic moiety accounted for five degrees of unsaturation, compound 1 should be a tetracyclic molecule.
In the COSY spectrum, three fragments were easily deduced according to the correlations of H-3/H-2/H-1, H-5/H-6/H-7, and H-9/H-11/H-21. In the HMBC spectrum, the longrange cross peaks originated from Me-18, Me-20, and Me-22-Me-25 established a tetracyclic skeleton of 1. Further HMBC correlations of H-3 and H-3 ′ to the carboxyl groups at δ C 170.0 and 167.8 confirmed the attachment of the acetoxyl and methoxyl at C-3 and C-19, respectively. Accordingly, the planar structure of 1 was established as shown in Figure 2.
To further assign its absolute stereochemistry, the theoretical calculation of the electronic circular dichroism (ECD) was adopted. As shown in Figure 3, the calculated ECD of (3S,5S,8S,9S,10R,13R, 14R,16R)-1 (a) showed the same Cotton effects as those of the experimental ones. On the basis of the above evidence, 1 was therefore assigned and given the trivial name of Andrastone A.
Compound 2 was also obtained as colorless oil. The molecular formula was established as C 28 H 36 O 9 on the basis of the sodium adduct at m/z 539.2257 [M + Na] + (calcd for C 28 H 36 O 9 Na, 539.2252) in its HRESIMS spectrum. The 1 H and 13 C NMR spectra showed 28 carbons consisting of seven methyls, one methoxyl, three methylenes, five methines (one olefinic, two oxygenated, and two aliphatic), and 12 non-protonated carbons (two ketones, three carboxyls, one olefinic, one oxygenated, and five aliphatic). These signals were very similar to those of compound 3, a meroterpene that was established as citreohybriddione A by single X-ray crystallography (Figure 4) (Kosemura et al., 1991a). However, clear differences were found in 2 showing that C-16, Me-18 were upshifted up to 4.7 and 5.7 ppm, respectively, suggesting the hydroxy moiety at the C-16 position should be the β-configuration in 2, instead of α in 3. This was confirmed by the NOESY correlations of 3 ′ -Me to Me-18. By comparison of the experimental and theoretical calculation of the ECD spectra (Figure 3), 2 was therefore assigned to be 16-epi-citreohybriddione A.
To uncover its inhibition mechanism, 1 was first subjected to flow cytometry. According to a previous study, Paclitaxel can induce apoptosis via arresting cells mainly in the G2/M phase of the cell cycle (Jelinek et al., 2015;Maushagen et al., 2016). Therefore, it was used as the positive control. As shown in Figure 5, Paclitaxel and 1 could obviously induce apoptosis in HepG2 cells.
Since caspase is a key pathway to induce apoptosis (Porter and Janicke, 1999;Li and Yuan, 2008), we measured the activities of the caspase-3, 8, and 9. As a result, 1 could significantly increase the activities of caspase-3 and caspase-8, but had almost no effect on caspase-9 (Figure 6), suggesting that the apoptosis was caused by direct caspase-8-mediated caspase-3 activation (Kaufmann et al., 2008).
Moreover, a dual luciferase reporter gene assay was also carried out to investigate the RXRα transcriptional activity. Interestingly, 1 not only increased the reporter transcriptional activation of RXRα, but also reduced the transactivity of RXRα induced by 9-cis-RA (Figure 7).

CONCLUSIONS
In summary, from the deep-sea-derived fungus Penicillium alliisativi, two new and one known meroterpenes were obtained. Andrastone A (1) showed significant inhibitory effect selectively against HepG2 tumor cells by activating caspase-3 and regulating the transcriptional activation function of RXRα.

DATA AVAILABILITY STATEMENT
All datasets for this study are included in the manuscript/Supplementary Files.

ACKNOWLEDGMENTS
We thank Mr. Tian-Hua Zhong for the measurement of the NMR data.