Steroids from the Deep-Sea-Derived Fungus Penicillium granulatum MCCC 3A00475 Induced Apoptosis via Retinoid X Receptor (RXR)-α Pathway

Five new ergostanes, penicisteroids D−H (1−5), were isolated from the liquid culture of the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475, along with 27 known compounds. The structures of the new steroids were established mainly on the basis of extensive analysis of 1D and 2D NMR as well as HRESIMS data. Moreover, the absolute configurations of 1 were confirmed unambiguously by the single-crystal X-ray crystallography. Compounds 2 and 4–7 showed moderate antiproliferative effects selectively against 12 different cancer cell lines with IC50 values of around 5 μM. Compounds 2 and 6, potent RXRα binders with Kd values of 13.8 and 12.9 μM, respectively, could induce apoptosis by a Retinoid X Receptor (RXR)-α-dependent mechanism by regulating RXRα transcriptional expression and promoting the poly-ADP-ribose polymerase (PARP) cleavage. Moreover, they could inhibit proliferation by cell cycle arrest at the G0/G1 phase.


Results and Discussion
Compound 1 was isolated as colorless needle crystals. The sodium adduct molecular ion peak at m/z 479.3547 in the HRESIMS (High Resolution Electrospray Ionization Mass Spectroscopy) indicated its molecular formula as C30H48O3, requiring six degrees of unsaturation. The 1 H NMR spectrum ( Figure S1 Figure S2) revealed the presence of 30 carbons, including seven sp 3 methyls, seven methylenes, twelve methines (two oxygenated sp 3 and three sp 2 ), and four non-protonated carbons (two sp 2 and two sp 3 ). Altogether, the 1D NMR spectroscopic data (Tables 1 and 2) indicated one acetoxyl moiety [δH 1.98 (3H, s, Me-2'); δC 172.3 (s, C-1'), 21.7 (q, Me-2')] and a 28-carbon skeleton. Since four olefinic carbons and one carboxyl moiety accounted for three unsaturation degrees, 1 should be a tetracyclic molecule. The assumption, along with the presence of four methyl doublets and two methyl singlets, suggested a steroid skeleton for 1.
In the COSY (Correlation Spectroscopy) spectrum ( Figure S4), a long chain was constructed according to correlations from H-3 (δH 3.  Figure 2). Since the chemical shifts of the two sp 2 methine protons were overlapped at δH 5.19 (m), the above deduced two fragmentations could not be connected according to the COSY spectrum. However, it could be easily resolved by the HMBC (Heteronuclear Multiplebond Correlation) cross peaks of H-20, H-24, Me-21, and Me-28 ( Figure S5). Furthermore, the HMBC correlations of two methyls of Me-18 and Me-19 constructed four rings of the steroid skeleton. In addition, the connection of the acetoxyl moiety to C-16 was proved by the HMBC correlation of H-16 to the carboxyl carbon at δC 172. 3. On the basis of the above evidence, the planar structure of 1 was then assigned as 16-acetoxy-3-hydroxyergost-5,22E-diene, the 7-dehydroxy derivate of penicisteroid A [13].

Results and Discussion
Compound 1 was isolated as colorless needle crystals. The sodium adduct molecular ion peak at m/z 479.3547 in the HRESIMS (High Resolution Electrospray Ionization Mass Spectroscopy) indicated its molecular formula as C 30 Figure S2) revealed the presence of 30 carbons, including seven sp 3 methyls, seven methylenes, twelve methines (two oxygenated sp 3 and three sp 2 ), and four non-protonated carbons (two sp 2 and two sp 3 ). Altogether, the 1D NMR spectroscopic data (Tables 1 and 2) indicated one acetoxyl moiety [δ H 1.98 (3H, s, Me-2 ); δ C 172.3 (s, C-1 ), 21.7 (q, Me-2 )] and a 28-carbon skeleton. Since four olefinic carbons and one carboxyl moiety accounted for three unsaturation degrees, 1 should be a tetracyclic molecule. The assumption, along with the presence of four methyl doublets and two methyl singlets, suggested a steroid skeleton for 1.
In the COSY (Correlation Spectroscopy) spectrum ( Figure S4) Figure 2). Since the chemical shifts of the two sp 2 methine protons were overlapped at δ H 5.19 (m), the above deduced two fragmentations could not be connected according to the COSY spectrum. However, it could be easily resolved by the HMBC (Heteronuclear Multiple-bond Correlation) cross peaks of H-20, H-24, Me-21, and Me-28 ( Figure S5). Furthermore, the HMBC correlations of two methyls of Me-18 and Me-19 constructed four rings of the steroid skeleton. In addition, the connection of the acetoxyl moiety to C-16 was proved by the HMBC correlation of H-16 to the carboxyl carbon at δ C 172.3. On the basis of the above evidence, the planar structure of 1 was then assigned as 16-acetoxy-3-hydroxyergost-5,22E-diene, the 7-dehydroxy derivate of penicisteroid A [13].   (Figure 2). However, the terrible overlapped signals of H-9 (δH 0.96 m)/H-14 (δH 1.00 m)/H-15a (δH 1.02 m) and H-12a (δH 1.28 m)/H-17 (δH 1.29 m) make it impossible to establish its relative configuration. Fortunately, an orthorhombic crystal was obtained from MeOH. By the single X-ray diffraction analysis using Cu-Kα radiation(Tables S1-S4), the absolute configurations of C-3 and C-16 were both assigned as S (Figure  The relative configuration of 1 was established according to the coupling constants and NOESY (Nuclear Overhauser Effect) spectrum ( Figure S6). The chair conformations of rings A and B and a trans-relationship between them were established by the large half-peak-width of H-3 (W1/2 = 15.  The relative configuration of 1 was established according to the coupling constants and NOESY (Nuclear Overhauser Effect) spectrum ( Figure S6). The chair conformations of rings A and B and a trans-relationship between them were established by the large half-peak-width of H-3 (W1/2 = 15.  The relative configuration of 1 was established according to the coupling constants and NOESY (Nuclear Overhauser Effect) spectrum ( Figure S6). The chair conformations of rings A and B and a trans-relationship between them were established by the large half-peak-width of H-3 (W1/2 = 15. (δH 1.29 m) make it impossible to establish its relative configuration. Fortunately, an orthorhombic crystal was obtained from MeOH. By the single X-ray diffraction analysis using Cu-Kα radiation(Tables S1-S4), the absolute configurations of C-3 and C-16 were both assigned as S ( Figure   ) correlations of 1. The relative configuration of 1 was established according to the coupling constants and NOESY (Nuclear Overhauser Effect) spectrum ( Figure S6). The chair conformations of rings A and B and a trans-relationship between them were established by the large half-peak-width of H-3 (W1/2 = 15. 3). Accordingly, 1 was unambiguously established as 16β-acetoxy-3β-hydroxyergost-5,22E-diene and named penicisteroid D.  Compound 2 was isolated as an amorporous powder. The molecular formula, C30H48O5, was assigned by the sodium adduct molecular ion peak at m/z 511.3383 in the HRESIMS, indicating six degrees of unsaturation. The 1 H and 13 C NMR spectra ( Figures S7 and S8) exhibited 30 carbons, including four doublet and three singlet methyls, five methylenes, 14 methines (four oxygenated and three olefinic), and four non-protonated carbons (one olefinic and one carboxyls). These signals were closely similar to those of 1 except for two additional hydroxyls in 2. In the COSY spectrum ( Figure  S10), correlations were found of H-8 via the oxymethine proton at δH 3.75 to H-6 and via H-9 to another oxymethine proton at δH 4.27, suggesting connections of hydroxyls at C-7 and C-11 positions, respectively. Further confirmation was found by the HMBC correlations of H-7 to C-5/C-6/C-8/C-9 and H-11 to C-8/C-9/C-10 ( Figure S11). The large coupling constant of JH7,H8 (J = 7.3 Hz) was indicative of its axial α-orientation and the small coupling constants of JH9,H11;H11,H12 (J = 3.6, 2.9 Hz) pointed to its  Compound 2 was isolated as an amorporous powder. The molecular formula, C 30 H 48 O 5 , was assigned by the sodium adduct molecular ion peak at m/z 511.3383 in the HRESIMS, indicating six degrees of unsaturation. The 1 H and 13 C NMR spectra ( Figures S7 and S8) exhibited 30 carbons, including four doublet and three singlet methyls, five methylenes, 14 methines (four oxygenated and three olefinic), and four non-protonated carbons (one olefinic and one carboxyls). These signals were closely similar to those of 1 except for two additional hydroxyls in 2. In the COSY spectrum ( Figure S10), correlations were found of H-8 via the oxymethine proton at δ H 3.75 to H-6 and via H-9 to another oxymethine proton at δ H 4.27, suggesting connections of hydroxyls at C-7 and C-11 positions, respectively. Further confirmation was found by the HMBC correlations of H-7 to C-5/C-6/C-8/C-9 and H-11 to C-8/C-9/C-10 ( Figure S11). The large coupling constant of J H7,H8 (J = 7.3 Hz) was indicative of its axial α-orientation and the small coupling constants of J H9,H11;H11,H12 (J = 3.6, 2.9 Hz) pointed to its axial-orientation. This was confirmed by the NOESY correlations of H-9 to H-7/H-11, H-7 to H-14, and H-8 to Me-18/Me-19 ( Figure S12). Accordingly, 2 was established as 16β-acetoxy-3β,7β,11β-trihydroxyergost-5,22-diene and named penicisteroid E.
The molecular formula of 4 was determined as C 30 H 50 O 6 based on the sodium adduct ion peak in its HRESIMS spectrum. The 1 H and 13 C NMR spectroscopic data (Figures S19 and S20) of 4 were close similar to those of 3 except that the oxygenated methine (δ C 68.0) at the C-11 position in 3 was replaced by the methylene (δ C 22.2) in 4, suggesting the absence of the hydroxyl group at C-11. This assumption was evidenced by the significant upshift of C-12 from δ C 50.2 to δ C 41.3 and C-9 from δ C 48.4 to δ C 45.4. Further confirmation was obtained by the COSY correlations ( Figure S22) of H-12 (δ H 2.01 dt, J = 12.7, 3.1 Hz) to H-11 (δ H 1.42 m). Accordingly, 4 was defined as 16β-acetoxy-3β,5α,6β,7β-tetrahydroxyergost-22E-ene, and named penicisteroid G.
All 19 steroids (1-19) were tested for cytotoxicity against 12 cancer cell lines of SHG-44, HepG2, A549, BIU-87, BEL-7402, ECA-109, Hela-S3, PANC-1, SW620, HcT116, MCF-7, and MB-231. They showed selectively cytotoxicity against A549, BIU-87, BEL-7402, ECA-109, Hela-S3, and PANC-1 cells. However, none exhibited antiproliferative effect against SW620, HcT116, MCF-7, and MB-231 cancer cell lines (Table 3). Further investigation by flow cytometry (Figure 4) and the Western blotting ( Figure 5) indicated compounds 2 and 4-7 could induce apoptosis in A549 cells. Moreover, compounds 2 and 6 could also inhibit cell proliferation by cell cycle arresting at G0/G1 phase ( Figure 6).          To further investigate the apoptosis mechanisms, 2 and 6 were tested for transcriptional activities on two nuclear receptors, RXRα and Nur77 (also called NGFIB), using the dual-luciferase reporter gene assay. However, they did not display positive effects on Nur77. Instead, they could significantly decrease the transcriptional activation of RXRα induced by 9-cis ( Figure 7) in a dosedependent manner (Figure 8). It indicated that 2 and 6 might have selectivity effected transcriptional activation of nuclear receptors and only acted on RXRα. and the tested compounds (50 μM) for 18 h, respectively. The luciferase activity of the 9-cis group is normalized to 1. (Right) HEK293T cells transfected pBind-Nur77-LBD and PG5 plasmids were treated with the tested compounds (50 μM) for 24 h. The luciferase activity of the control group is normalized to 1. ### p < 0.001 versus control, ns: no significance, ** p < 0.01, *** p < 0.001 versus the 9cis group. To further investigate the apoptosis mechanisms, 2 and 6 were tested for transcriptional activities on two nuclear receptors, RXRα and Nur77 (also called NGFIB), using the dual-luciferase reporter gene assay. However, they did not display positive effects on Nur77. Instead, they could significantly decrease the transcriptional activation of RXRα induced by 9-cis ( Figure 7) in a dose-dependent manner ( Figure 8). It indicated that 2 and 6 might have selectivity effected transcriptional activation of nuclear receptors and only acted on RXRα.  To further investigate the apoptosis mechanisms, 2 and 6 were tested for transcriptional activities on two nuclear receptors, RXRα and Nur77 (also called NGFIB), using the dual-luciferase reporter gene assay. However, they did not display positive effects on Nur77. Instead, they could significantly decrease the transcriptional activation of RXRα induced by 9-cis ( Figure 7) in a dosedependent manner (Figure 8). It indicated that 2 and 6 might have selectivity effected transcriptional activation of nuclear receptors and only acted on RXRα.  Furthermore, we used fluorescence quenching assay ( Figure 9) to analyze compounds for binding to RXRα-LBD and identified compounds 2 and 6 as a potent binder with a Kd of 13.8 μM and 12.9 μM, and the role of RXRα was illustrated by data showing that transfection of RXRα small interfering RNA (si-RNA), which inhibited RXRα expression, abrogated the apoptosis effect induced by compounds 2 and 6 ( Figure 10). The above findings indicated that these compounds might have a useful impact on RXRα-mediated growth inhibition and apoptosis induction in cancer cells as well as RXRα-dependent regulation of gene expression.   The luciferase activity of the 9-cis group is normalized to 1. ### p < 0.001 versus control, * p < 0.05, ** p < 0.01, *** p < 0.001 versus the 9-cis group.
Furthermore, we used fluorescence quenching assay ( Figure 9) to analyze compounds for binding to RXRα-LBD and identified compounds 2 and 6 as a potent binder with a Kd of 13.8 µM and 12.9 µM, and the role of RXRα was illustrated by data showing that transfection of RXRα small interfering RNA (si-RNA), which inhibited RXRα expression, abrogated the apoptosis effect induced by compounds 2 and 6 ( Figure 10). The above findings indicated that these compounds might have a useful impact on RXRα-mediated growth inhibition and apoptosis induction in cancer cells as well as RXRα-dependent regulation of gene expression. Furthermore, we used fluorescence quenching assay ( Figure 9) to analyze compounds for binding to RXRα-LBD and identified compounds 2 and 6 as a potent binder with a Kd of 13.8 μM and 12.9 μM, and the role of RXRα was illustrated by data showing that transfection of RXRα small interfering RNA (si-RNA), which inhibited RXRα expression, abrogated the apoptosis effect induced by compounds 2 and 6 ( Figure 10). The above findings indicated that these compounds might have a useful impact on RXRα-mediated growth inhibition and apoptosis induction in cancer cells as well as RXRα-dependent regulation of gene expression.   Furthermore, we used fluorescence quenching assay ( Figure 9) to analyze compounds for binding to RXRα-LBD and identified compounds 2 and 6 as a potent binder with a Kd of 13.8 μM and 12.9 μM, and the role of RXRα was illustrated by data showing that transfection of RXRα small interfering RNA (si-RNA), which inhibited RXRα expression, abrogated the apoptosis effect induced by compounds 2 and 6 ( Figure 10). The above findings indicated that these compounds might have a useful impact on RXRα-mediated growth inhibition and apoptosis induction in cancer cells as well as RXRα-dependent regulation of gene expression.

General Experimental Procedures
Optical rotations were recorded on an MCP 500 automatic polarimeter (Anton Paar Trading Co. Ltd., Shanghai, China) under 20 • C. Ultraviolet spectra were detected by a UV8000 UV/Vis spectrophotometer (Shanghai Metash instrument Co., Ltd., Shanghai, China). The HRESIMS spectra were measured by a Xevo G2 Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). The NMR spectra were recorded on a 400 MHz spectrometer (Bruker, Fällanden, Switzerland) using TMS as the internal standard. Reversed-phase HPLC was performed on a 1260 infinity instrument (Agilent Technologies, San Diego, CA, USA) equipped with the DAD detector. Purifications by column chromatography (CC) were performed on silica gel, Sephadex LH-20, and ODS. The TLC plates were visualized under UV light or by spraying with 10% H 2 SO 4 .

Fungal Identification, Fermentation, and Extract
The fungus of Penicillium granulatum MCCC 3A00475 was provided by the Marine Culture Collection of China (MCCC). The isolation and identification were described previously [11,12].
Erlenmeyer flasks (250 mL) containing 100 mL fermentation media were directly inoculated with a quarter plate of the strain spores. After 2 days of incubation at 28 • C on a rotary shaker at 180 r/min, 20 mL seed cultures were transferred into a total of 100 flasks (1 L) containing 380 mL of defined medium (10 g glucose, 20 mL mannitol, 5 g potato peptone, 5 g monosodium glutamate, 3 g yeast extract, 3 g maltose dissolved in 1 L of water, pH 7.5). The flasks were cultured with shaking at 180 rpm and 28 • C for 11 days.

Cell Proliferation Assay
Cytotoxic activities of all steroids were conducted on human glioma cell line SHG-44, liver cancer cell lines HepG2, and 7402, non-small cell lung cancer cell line A549, bladder cancer cell line BIU-87, esophageal cancer cell line ECA-109, cervix cancer cell line Hela-S3, pancreatic cancer cell line PANC-1, colon carcinoma cell lines SW620 and HcT116, breast cancer cell lines MCF-7and MB-231 by MTT method as reported previously [32].

Apoptosis Determination by FCM
The levels of cellular superoxide were assessed by using FITC Annexin V Apoptosis Detection Kit (556547, BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, A549 cells treated with trypsin and resuspended in Annexin V binding buffer, then the cells were labelled by PI and FITC. At last, fluorescence was measured by flow cytometry using FITC-A (CytoFLEX, Beckman Coulter, Kraemer Boulevard Brea, CA, USA).