Unprecedented Monoterpenoid Polyprenylated Acylphloroglucinols with a Rare 6/6/5/4 Tetracyclic Core, Enhanced MCF-7 Cells’ Sensitivity to Camptothecin by Inhibiting the DNA Damage Response

(±)-Hypersines A–C (1–3), the three pairs of enantiomerically pure monoterpenoid polyprenylated acylphloroglucinols with an unprecedented 6/6/5/4 fused ring system, were isolated from Hypericum elodeoides. Their structures, including absolute configurations, were elucidated by comprehensive spectroscopic data, single-crystal X-ray diffraction, and quantum chemical calculations. The plausible, biosynthetic pathway of 1–3 was proposed. Moreover, the bioactivity evaluation indicated that 1a might be a novel DNA damage response inhibitor, and could enhance MCF-7 cell sensitivity to the anticancer agent, camptothecin.


Plant Material
The Environment and Ecology, Xiamen University), and a voucher specimen (ID HE201608) deposited at the School of Pharmaceutical Sciences, Xiamen University.

Quantum Chemical Calculation of 13 C NMR
All structures were optimized at the b3lyp/6-31+g (d, p) level for all conformations using Gaussian09 [25]. The 13 C NMR shielding constants were computed using the Gauge Independent Atomic Orbital (GIAO) method [26] at the mpw1pw91/6-311+g (2d, p) level, with SMD in chloroform using Gaussian09. The final, calculated 13 C NMR chemical shifts were obtained by averaging the 13 C NMR chemical shifts of the optimized conformers according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G).

Quantum Chemical Calculation of ECD Spectra
Monte Carlo conformational searches were carried out by means of the Spartan's 10 software, using the Merck Molecular Force Field (MMFF). The conformers with a Boltzmann population of over 2% were chosen for ECD calculations, and were then initially optimized at B3LYP/6-311g (2d, p) level in MeOH using the CPCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in MeOH using time-dependent density-functional theory (TDDFT) at the B3LYP/6-311g (2d, p) level, for all conformers. The rotatory strengths for 60 excited states were calculated. ECD spectra were generated using the SpecDis 1.7.1 program (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, CA, USA) from dipole-length rotational strengths, and by applying Gaussian band shapes with sigma = 0.3 eV.

Cell Culture
The MCF-7 cell line was obtained from the National Collection of Authenticated cell cultures (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, BasalMedia, Shanghai, China), containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C, and 5% CO 2 .

MTT Assay
The cell viability was evaluated by the MTT assay. MCF-7 cells were seeded in a 96-well plate, at a density of 1 × 10 4 cells/well. The cells were grown overnight at 37 • C with humidified 5% CO 2 , and then treated with compounds 1a-3b (20 µM) for 48 h. The supernatants were discarded and 15 µL MTT reagent, as well as 60 µL DMEM, were added. After incubating for 4 h, the supernatants were removed and DMSO was added. The absorbances were measured at 490 nm by the Thermo Multiskan MK3 (Thermo Scientific, Waltham, MA, USA). The cell viability was analyzed as follows:

Western Blotting
Western blotting was conducted as described [27]. After exposure to compounds and CPT for 48 h, cells were lysed by the RIPA buffer with a protease inhibitor and a phosphatase inhibitor (MCE). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into the PVDF membrane. The membranes were blocked in no-fat milk with TBST and incubated with primary antibody at 4 • C overnight. Then, the membranes were washed 3 times in TBST and incubated with secondary antibody at room temperature. After being washed, the target proteins on the membranes were detected by chemi-luminescence reagent (Advansta, San Jose, CA, USA).

Cell Cycle Analysis
The MCF-7 cells were seeded in 6 cm dishes. After exposure to CPT with or without compound 1a for 48 h, the cells were harvested by trypsin with no EDTA, and fixed in cold 75% ethanol overnight. Following centrifuge (1000 rpm, 5 min room temperature) and removing the supernatant, the cells were incubated with 10 µg/mL RNase in PBS at 37 • C for 30 min, and with PI staining buffer at room temperature for 5 min in the dark. The cell cycle distribution was analyzed by flow cytometry (CytoFlex, Beckman Coulter, Los Angeles, CA, USA).

Statistical Analysis
The data were shown as mean ± SD, and the differences between samples were evaluated statistically with ANOVA analysis and Tukey's posteriori comparison through GraphPad Prism 6.0. Differences were statistically significant at p < 0.05.

Isolation of Compounds 1-3
The whole plants of H. elodeoides were extracted with methanol at room temperature. The crude extract was subjected to the silica gel column chromatography, eluting with CH 2 Cl 2 , EtOAc, and methanol. Compounds 1-3 were isolated from the CH 2 Cl 2 fraction by a series of column chromatography. Then, three pairs of enantiomers, (±)-Hypersines A-C, were purified by chiral HPLC separation with CHIRALPAK ® IG column.

Structural Elucidation of Compounds 1−3
The structures, including absolute configurations of isolated new compounds (±)hypersines A-C, were elucidated by comprehensive spectroscopic data, single-crystal X-ray diffraction, and quantum chemical calculations.

Structural Elucidation of Compounds 1−3
The structures, including absolute configurations of isolated new compounds (±)hypersines A-C, were elucidated by comprehensive spectroscopic data, single-crystal Xray diffraction, and quantum chemical calculations.

Plausible Biosynthetic Pathway to 1−3
(±)-Hypersines A-C represent the first example of MTPAPs with a rare 6/6/5/4 ring system, which is quite different from the known MTPAPs. The plausible biosynthetic pathway for 1-3 was proposed (Scheme 1). Acylphloroglucinol was considered as the biogenetic precursor to PPAPs, which had undergone methylation reaction to obtain intermediate I with a filicinic acid core. Subsequently, intermediate II could be formed by the

Plausible Biosynthetic Pathway to 1-3
(±)-Hypersines A-C represent the first example of MTPAPs with a rare 6/6/5/4 ring system, which is quite different from the known MTPAPs. The plausible biosynthetic pathway for 1-3 was proposed (Scheme 1). Acylphloroglucinol was considered as the biogenetic precursor to PPAPs, which had undergone methylation reaction to obtain intermediate I with a filicinic acid core. Subsequently, intermediate II could be formed by the enzyme-catalyzed addition of geranyl pyrophosphate to the filicinic core. Then, epoxidation occurred at ∆ [8] to generate an epoxypropane intermediate III. Next, the sixmembered oxygen ring was formed and generated intermediate IV. After the dehydration of OH-8, the unsaturated pyranoid ring was generated. Finally, the 6/6/5/4 tetracyclic skeleton was connected by [2 + 2] cycloadditions [9,19].

Biological Activity Evaluation of the Isolated Compounds
The biological activities of the isolated compounds, (±)-Hypersines A-C (1a-3b), were evaluated. In our previous studies, several PPAPs were found to show an inhibitory effects on the proliferation of human breast cancer cells (MCF-7) [6,21]. In this study, the MTT assay was used to measure the viability of MCF-7 cells after exposure to the isolated compounds. The results showed that none of the compounds could suppress the proliferation of MCF-7 cells in 20 μM ( Figure 6A). Poly ADP-ribose polymerase (PARP) is a marker protein of apoptosis. Interestingly, when the compounds were co-treated with the anticancer agent, camptothecin (CPT), in a low concentration, 1a (20 μM) could visibly trigger the cleavage of PARP combined with CPT (50 nM), while there was no PARP cleavage when treated with CPT (50 nM) alone ( Figure 6B). As known, CPT causes cell death by suppressing DNA synthesis and eliciting DNA double-strand break (DSB) [31]. The above result indicated that 1a could reinforce the CPT-induced apoptosis of MCF-7 cells. Further research exhibited that the percentage of MCF-7 cells in the G2/M phase increased after the co-treatment of CPT and 1a ( Figure 6C or Figure 6D). Meanwhile, the combination of CPT and 1a obviously augmented the expression of γ-H2AX (the biomarker of DSB) ( Figure 6E) [32]. Furthermore, the CPT restrained the phosphorylation of the Ser 10 of histone H3 (pS10-H3, the mitotic biomarker) [33]. This may be owing to CPT-induced DSB causing DNA damage response (DDR), which means that cells with damaged DNA cannot prematurely undergo mitosis [34]. However, synergy between CPT and 1a could strongly elevate the level of pS10-H3, indicating that the majority of cells might enter M phase, which is demonstrated by the increasing level of Cyclin B1 ( Figure 6E) [33]. Furthermore, the flow cytometry analysis indicated that the cells were arrested at G2/M phase with the co-treatment of CPT and 1a (Figures 6C or Figure 6D). The above data indicated Scheme 1. Plausible Biosynthetic Pathway to 1-3.

Biological Activity Evaluation of the Isolated Compounds
The biological activities of the isolated compounds, (±)-Hypersines A-C (1a-3b), were evaluated. In our previous studies, several PPAPs were found to show an inhibitory effects on the proliferation of human breast cancer cells (MCF-7) [6,21]. In this study, the MTT assay was used to measure the viability of MCF-7 cells after exposure to the isolated compounds. The results showed that none of the compounds could suppress the proliferation of MCF-7 cells in 20 µM ( Figure 6A). Poly ADP-ribose polymerase (PARP) is a marker protein of apoptosis. Interestingly, when the compounds were co-treated with the anticancer agent, camptothecin (CPT), in a low concentration, 1a (20 µM) could visibly trigger the cleavage of PARP combined with CPT (50 nM), while there was no PARP cleavage when treated with CPT (50 nM) alone ( Figure 6B). As known, CPT causes cell death by suppressing DNA synthesis and eliciting DNA double-strand break (DSB) [31]. The above result indicated that 1a could reinforce the CPT-induced apoptosis of MCF-7 cells. Further research exhibited that the percentage of MCF-7 cells in the G2/M phase increased after the co-treatment of CPT and 1a ( Figure 6C or Figure 6D). Meanwhile, the combination of CPT and 1a obviously augmented the expression of γ-H2AX (the biomarker of DSB) ( Figure 6E) [32]. Furthermore, the CPT restrained the phosphorylation of the Ser 10 of histone H3 (pS10-H3, the mitotic biomarker) [33]. This may be owing to CPT-induced DSB causing DNA damage response (DDR), which means that cells with damaged DNA cannot prematurely undergo mitosis [34]. However, synergy between CPT and 1a could strongly elevate the level of pS10-H3, indicating that the majority of cells might enter M phase, which is demonstrated by the increasing level of Cyclin B1 ( Figure 6E) [33]. Furthermore, the flow cytometry analysis indicated that the cells were arrested at G2/M phase with the co-treatment of CPT and 1a ( Figure 6C or Figure 6D). The above data indicated that 1a might urge MCF-7 cells with CPT-induced DSB to progress into mitosis by restricting DDR and by rendering damaged DNA to remain in M phase. Compromised DNA entering into mitosis can lead to the apoptosis of cells [35]. This may be the reason why 1a potentiated the CPT-induced apoptosis of MCF-7 cells, but specific mechanisms remained to be investigated. DDR inhibitors are usually used as sensitizers of antitumor agents to play a role in tumor treatment [36]. Therefore, 1a might be considered as a novel inhibitor of DDR, and could enhance the sensitivity of MCF-7 cells to CPT. that 1a might urge MCF-7 cells with CPT-induced DSB to progress into mitosis by restricting DDR and by rendering damaged DNA to remain in M phase. Compromised DNA entering into mitosis can lead to the apoptosis of cells [35]. This may be the reason why 1a potentiated the CPT-induced apoptosis of MCF-7 cells, but specific mechanisms remained to be investigated. DDR inhibitors are usually used as sensitizers of antitumor agents to play a role in tumor treatment [36]. Therefore, 1a might be considered as a novel inhibitor of DDR, and could enhance the sensitivity of MCF-7 cells to CPT. The group for con means MCF-7 cells only exposed to DMSO. ** p < 0.01vs Con group. (E) MCF-7 cells were treated with CPT and 1a in combination for 48 h, the levels of relative protein (p10-H3, cyclin B1, γ-H2AX) were investigated by western blotting.

Conclusions
In summary, (±)-hypersines A-C (1-3), the three pairs of unprecedented MPAPs with a rare 6/6/5/4 tetracyclic scaffold, were obtained from H. elodeoides. As far as we know, hypersines A-C are the first reported MTPAPs characterized by a peculiar 6/6/5/4 tetracyclic core, formed by a rare, tensional four-membered carbocycle concurrently fused with five-and six-membered rings. Furthermore, the possible biosynthetic pathway of 1-3 was proposed, which might provide a reference for the organic synthesis of these compounds. The biological activity results showed that the combination of 1a and a low concentration of CPT could visibly trigger the cleavage of PARP, and could up-regulate the expression level of γ-H2AX, pS10-H3, and Cyclin B1, indicating that 1a might enhance MCF-7 cell sensitivity to camptothecin by inhibiting the DNA damage response. This is the first report of MTPAPs as potential sensitizers of anti-tumor drugs. In general, this study enriches the structural diversity and biological activity of MTPAPs, and provides novel insight for further understanding MTPAPs. However, among these compounds, only 1a exhibited potent activity. In future research, more analogues need to be obtained to further investigate the structure-activity relationship. A more detailed mechanism that 1a enhances MCF-7 cell sensitivity to CPT needs to be further explored. This research suggests Figure 6. (A) MCF-7 cells were exposed to compounds (1a-3b) for 48 h. The cells viability was measured via MTT assay. The group for con means MCF-7 cells only treated with DMSO. (B) MCF-7 cells were incubated with 50 nM CPT, with or without 20 µM compounds, for 48 h, western blotting was used to analyze the level of cleaved-PARP and PARP. (C) After treatment with CPT plus 1a for 48 h, the cell cycle of MCF-7 cells was determined through flow cytometry. (D) The percentages of G2/M phase. The group for con means MCF-7 cells only exposed to DMSO. ** p < 0.01vs Con group. (E) MCF-7 cells were treated with CPT and 1a in combination for 48 h, the levels of relative protein (p10-H3, cyclin B1, γ-H2AX) were investigated by western blotting.

Conclusions
In summary, (±)-hypersines A-C (1-3), the three pairs of unprecedented MPAPs with a rare 6/6/5/4 tetracyclic scaffold, were obtained from H. elodeoides. As far as we know, hypersines A-C are the first reported MTPAPs characterized by a peculiar 6/6/5/4 tetracyclic core, formed by a rare, tensional four-membered carbocycle concurrently fused with five-and six-membered rings. Furthermore, the possible biosynthetic pathway of 1-3 was proposed, which might provide a reference for the organic synthesis of these compounds. The biological activity results showed that the combination of 1a and a low concentration of CPT could visibly trigger the cleavage of PARP, and could up-regulate the expression level of γ-H2AX, pS10-H3, and Cyclin B1, indicating that 1a might enhance MCF-7 cell sensitivity to camptothecin by inhibiting the DNA damage response. This is the first report of MTPAPs as potential sensitizers of anti-tumor drugs. In general, this study enriches the structural diversity and biological activity of MTPAPs, and provides novel insight for further understanding MTPAPs. However, among these compounds, only 1a exhibited potent activity. In future research, more analogues need to be obtained to further investigate the structure-activity relationship. A more detailed mechanism that 1a enhances MCF-7 cell sensitivity to CPT needs to be further explored. This research suggests that the co-administration of 1a and chemotherapeutic drugs could be an effective strategy for overcoming tumor MDR, and this deserves further exploration.