Meroterpenoids From Ganoderma lucidum Mushrooms and Their Biological Roles in Insulin Resistance and Triple-Negative Breast Cancer

Ganoderma fungi as popular raw materials of numerous functional foods have been extensively investigated. In this study, five pairs of meroterpenoid enantiomers beyond well-known triterpenoids and polysaccharides, dayaolingzhiols I−M (1–5), were characterized from Ganoderma lucidum. Their structures were identified using spectroscopic and computational methods. Structurally, compound 1 features a novel dioxabicyclo[2.2.2]octan-3-one motif in the side chain. Ethnoknowledge-derived biological evaluation found that (+)-5 could activate Akt and AMPK phosphorylation in insulin-stimulated C2C12 cells, and (+)-5 could activate glucose uptake dose dependently in C2C12 cells. Furthermore, we found that (+)-1 (+)-4, and (–)-4 could significantly inhibit cell migration of the MDA-MB-231 cell line, of which (+)-4 showed significant inhibitory effects against cell migration of the MDA-MB-231 cell line in a dose-dependent manner. These findings revealed the meroterpenoidal composition of G. lucidum and its roles in the prevention of chronic diseases such as diabetes mellitus and triple-negative breast cancer.


INTRODUCTION
Insulin resistance (IR) is defined as an impaired biological response to insulin stimulation of target tissues mainly including the liver, muscle, and adipose tissue. Insulin resistance syndrome, also known as metabolic syndrome, is associated with a broad spectrum of diseases such as obesity, hyperglycemia, hypertension, dyslipidemia, non-alcoholic fatty liver disease, cardiovascular disease, polycystic ovarian syndrome, and type 2 diabetes mellitus (Allahbadia and Merchant, 2008;Willard et al., 2016;Cortés-Rojo et al., 2020;García-Carretero et al., 2021). Insulin resistance is mainly related to excess body fat and also genetic causes, which affects as many as one in three Americans and becomes a tremendous burden for the healthcare system of the United States (Balasubramanyam, 2006;McCarthy, 2014;Sharma et al., 2019;Rajesh and Sarkar, 2021). Now, the incidence of insulin resistance is growing at an alarming speed, and it is actually becoming a non-ignorable public concern worldwide. Accumulating evidence revealed that the inactivation of Akt and the activation of Foxo1 via inhibiting insulin receptor substrate 1 (IRS1) and insulin receptor substrate 2 (IRS2) might act as the underlying mechanism of metabolic syndromes (Guo, 2014). Targeting the pivotal molecules such as IRS or Akt or Foxo1 in the signaling cascade will therefore be a precise strategy for the intervention of insulin resistance-related diseases (Guo, 2014).
Breast cancer is still the most common cancer among women (Chowdhury et al., 2021;Loibl et al., 2021). The incidence rate accounts for about 30%, and the mortality rate is as high as 15% (Loibl et al., 2021). Triple-negative breast cancer (TNBC) is one of the subgroups of breast cancer which has an incidence of 15-20% and many characteristics such as invasive, resistant, and rapid growth rate Chowdhury et al., 2021). Due to its aggressive behavior, metastasis usually occurs in the liver, lungs, and brain Chowdhury et al., 2021). Discovering some potential molecules which could inhibit cell migration of TNBC may provide some enlightenment for its treatment.
Ganoderma fungi, well-known mushrooms of numerous functional foods, have received long-term attention worldwide. For example, 28785 documents are related to Ganoderma when searched using SciFinder till August 29, 2021. The taste and functional components of the Ganoderma fungus lead to its applications as food additives, raw materials, dietary supplements and remedies, even in Western countries (Leskosek-Cukalovic et al., 2010;Veljović et al., 2019;Krobthonga and Yingchutrakul, 2020). Polysaccharides and triterpenoids have long been considered the major chemical compositional elements of Ganoderma, whereas the other chemical composition remains largely unknown. In recent years, a number of meroterpenoids in Ganoderma have been reported (Jiang et al., 2021), representing a new research trend for Ganoderma metabolites. Phenol and terpene moieties are considered biologically active, and Ganoderma meroterpenoids possess both groups (Lu et al., 2007;Tan et al., 2018). We have focused on the investigation of Ganoderma meroterpenoids since 2009. As a result, many structurally intriguing and biologically important meroterpenoids have been characterized by us (Yan et al., 2019;Dai et al., 2020;Qin et al., 2020;Zhang et al., 2020). Due to considerable economic values, Ganoderma species have been cultivated in several places of our country. The concept of "one strain many compounds (OSMAC)" has been widely accepted in the related field. Ganoderma belongs to a higher fungal family; hence, it is necessary to gain an insight into the chemical profiling of Ganoderma meroterpenoids of Ganoderma lucidum produced in different locations, despite that much work has been carried out on this fungal species. G. lucidum, cultivated at Dayao County of Yunnan Province, is locally dictated to have distinct effects on diabetes, which thus inspired our interest. G. lucidum was reported to have antitumor effects, and triterpenoids are generally regarded as its antitumor ingredients. Given the increasing number of Ganoderma meroterpenoids being discovered, it is worth being concerned whether they have a role in tumor treatment. As a consequence, this study afforded five pairs of meroterpenoid enantiomers, dayaolingzhiol I−M (1-5), with biological activities toward insulin resistance and metastasis of TNBC. Herein, we describe their isolation, structure characterization, and biological evaluation.
Dayaolingzhiol J (2) was obtained as yellowish gums. It has a molecular formula C 22 H 30 O 5 deduced by its HRESIMS, 13 C NMR, and DEPT spectra. The 1 H NMR spectrum of 2 (Table 1) shows a typical ABX spin system [(δ H 6.92, d, J 2.5 Hz, H-3; δ H 6.72, dd, J 8.8, 2.5 Hz, H-5; and δ H 7.18, d, J 8.8 Hz, H-6)]. The 13 C NMR and DEPT spectra show four methyl (one oxygenated), four methylene, six methine (five sp 2 and one sp 3 ), and eight non-protonated carbons (one carbonyl and three oxygenated quaternary carbons). These data are similar to those of ganofuran B (Adams et al., 2010). The only difference is that a methoxy group appears at C-7 in 2 supported by the HMBC correlations ( Figure 2) of -OCH 3 (δ H 3.90)/C-7 (δ C 141.1). The significant ROESY correlation of H 2 -11 (δ H 2.00)/H 3 -20 (δ H 1.52) indicates that the Δ 12(13) double bond is E form. Compound 2 was isolated as a racemic mixture, and chiral HPLC was used to afford (+)-2 and (-)-2. In general, it is a great challenge to assign the absolute configuration at C-9 in the side chain. In this case, computational methods such as ECD calculations at the APFD/ 6-311+g(2d,p) and B3LYP/6-31(d,p) level were carried out ( Figure 4 and Supplementary Figure S52). The results reveal  3.90 s 61.5 q 1-OH a 11.06 s 4-OH a 9.14 s a These signals are observed in DMSO-d 6 .
Dayaolingzhiol L (4) was obtained as yellowish gums, and its molecular formula was assigned as C 22 H 30 O 5 by analysis of its HRESIMS, 13 C NMR, and DEPT spectra. The 1 H NMR spectrum of 4 ( Table 2) shows a typical ABX spin system (δ H 7.25, d, J 2.9 Hz, H-3; δ H 7.01, dd, J 8.9, 2.9 Hz, H-5; δ H 6.79, d, J 8.9 Hz, H-6), suggesting the presence of a 1,2,4-trisubstituted benzene ring. The 13 C NMR and DEPT spectra show four methyl (one oxygenated), five methylene, six methine (five sp 2 and one sp 3 ), and seven non-protonated carbons (one ketone and one   carbonyl). These data are similar to those of fornicin C (Niu et al., 2006). The only difference is a methyl ester in 4, instead of the free carboxyl group in fornicin C, supported by the HMBC correlations (Figure 2) of -OCH 3 (δ H 3.68)/C-7 (δ C 177.7). The ROESY correlation of H 2 -12 (δ H 2.08)/H 3 -15 (δ H 1.61) indicates that the Δ 13(14) double bond is E form. Compound 4 is also a racemic mixture, and the absolute configurations of the two enantiomers after chiral separation by HPLC were assigned as R for (+)-4 and S for (-)-4, respectively, by comparison of their CD spectra with those of (+)− and (-)−applanatumol S . The structure of 4 was thus determined and named dayaolingzhiol L.
In this experiment, we obtained five long-chain meroterpenoids dayaolingzhiols I−M (1-5). Among them, dayaolingzhiol I (1) is a relatively rare meroterpenoid with dioxabicyclo[2.2.2]octan-3-one motif of G. lucidum. At the same time, dayaolingzhiol J (2) belongs to the benzofuran type of meroterpenoid, and few benzofuran type of meroterpenoids have been reported from Ganoderma so far (Adams et al., 2010). These two pairs of meroterpenoids together with the remaining three pairs of new meroterpenoids reveal the structural diversity of Ganoderma meroterpenoids.
Ganoderma lucidum cultivated at Dayao County of Yunnan Province is locally used as a health-care product with a pronounced effect on diabetes. To explore the protective effect of Ganoderma meroterpenoids against IR, insulin exposed C2C12 myotubes were used. C2C12 cells were pretreated with different compounds for 24 h, and cell viability was not decreased at 20 μM. The CCK-8 assay showed no obvious cytotoxicity of compounds, except (+)-4 and (-)-4 (Supplementary Figure  S54). When the cells differentiated into myotubes, they were pretreated with 100 nM insulin for 24 h to mimic the IR model and then exposed to 20 μM compounds for an additional 24 h. In the cellular model of IR, insulin-stimulated glucose uptake and the expression of phospho-adenosine monophosphate-activated protein kinase (p-AMPK) and phospho-AKT (p-AKT) were lower than those in control cells. The results showed that incubation with (+)-1, (-)-4, and (-)-5 caused an increase in the phosphorylation of AMPK in IR C2C12 cells after insulin stimulation. At the same time, compound (+)-5 treatment caused an increase in protein expression of p-AMPK and p-AKT in IR C2C12 cells ( Figure 6A−C). Further glucose uptake experiments showed that compound (+)-5 improved the capacity of glucose uptake in IR cells in a concentration-dependent manner ( Figure 6D). So far, some meroterpenoids were found to have beneficial effects to insulin sensitivity in PA-induced C2C12 cells, such as ganomycin C and ganodercin D . Ganomycin I exhibited potent insulin-sensitizing effects in KK-A y mice, and its analog (R,E)-5-(4-(tert-butyl)phenyl)-3-(4,8-dimethylnona-3,7-dien-1-yl)furan-2(5H)-one ameliorates insulin resistance (Wang et al., 2017;Wang et al., 2018). These meroterpenoids with long side chains showed advantages in the amelioration of insulin resistance.
Our previous study showed that Ganoderma meroterpenoids with long side chains are active toward breast cancer cell migration . In this article, 10 optically active compounds, all possessing a long side chain, were tested for their suppressive activity in the triple-negative breast cancer cell line (MDA-MB-231) by using cell viability and cell migration assays. First, the cell viability assay was carried out in the MDA-MB-231 cell line. As shown in Figure 7A, compounds demonstrated weak effect on cell viability at the concentration of 20 μM. Wound healing assay was investigated in the MDA-MB-231 cell line at 20 μM. The results showed that compounds (+)-1, (+)-4, and (-)-4 exhibit a migration inhibitory effect compared to the DMSO group in the MDA-MB-231 cell line, of which (+)-4 showed stronger inhibitory effect ( Figures 7B, D). Furthermore, the effect of compound (+)-4 was found to be dose-dependent at concentrations of 10 μM, 20 μM, and 30 μM (Figures 7C,E).

Extraction and Isolation
The powdered fruiting bodies of G. lucidum (30.0 kg) were extracted with 95% EtOH under percolation (240 L) at room temperature, and a crude extract (2.1 kg) was provided and then was suspended in H 2 O and partitioned with EtOAc three times to obtain an EtOAc part (1.1 kg). This extract was separated by an MCI gel CHP 20P column (MeOH/H 2 O, 40-100%) to afford thirteen fractions (Fr.1-Fr.13).

Insulin Resistance Assay
Cell Culture C2C12 cells, a mouse skeletal muscle myoblast line (Procell Life Science and Technology Co., Wuhan, China), were cultured by using the same method as previously described . When the cells differentiated into elongated, multinucleated myotubes, they were exposed to 100 nM insulin for an additional 24 h to mimic insulin resistance.
Cell Viability Assay C2C12 (5× 10 5 cells/mL) were seeded into 96-well plates with complete DMEM. After being cultured overnight, C2C12 cells were treated with five pairs of meroterpenoids or DMSO for 24 h. Then Cell Count Kit-8 (CCK-8, Beyotime, Shanghai, China) was added into each well for 1 h at 37°C. The absorbance of each well was recorded at 450 nm using a microplate reader (BioTek, United States).

Glucose Uptake Assay
C2C12 cells were incubated in DMEM (low glucose) for 6 h and placed in high-glucose DMEM before adding compounds, and 30 min after compound treatment, insulin (100 nmol/L) was added and cultured for 4 h. Glucose content of the culture supernatant was measured using the Glucose Colorimetric/ Fluorometric Assay Kit (BioVison, K606-100).

Western Blot
After different compound treatment for 24 h in the IR model, total protein was extracted from the cell lines using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) containing protease cocktail (Roche, Germany) and quantified the protein samples using the BCA assay (Thermo Scientific, United States). Equal amounts of protein extracts were separated by 8% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA, then with the indicated antibodies overnight at 4°C, and followed the incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. The bands were visualized and measured using the ECL kit (Pierce, United States). The densitometry analysis of the immunoblot results was performed using ImageJ software (NIH, United States).

Statistical Analysis
All experimental data in this study were performed in triplicate. The results were represented as mean ± SD. Statistical analyses were performed using Graphpad Prism 6 (GraphPad Software, San Diego, CA, United States) with one-way ANOVA. Differences were considered significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Wound Healing Assay in MDA-MB-231 Cells
The biological evaluation for wound healing in MDA-MB-231 cells was conducted as the previously reported protocols .

CONCLUSION
To conclude, triterpenoids and polysaccharides as chemical compositions of Ganoderma mushrooms have been traditional gnosia for decades. Our present study resulted in the isolation of five pairs of meroterpenoidal enantiomers, representing new chemical composition of G. lucidum and aiding an in-depth insight into Ganoderma metabolites. The activation of (+)-5 on Akt, AMPK, and glucose uptake in C2C12 cells indicates its role in diabetes prevention. In addition, the inhibitory effects of compounds (+)-1, (+)-4, and (-)-4 on cell mobility in MDA-MB-231 cells indicate their role in TNBC. These results shed light on the traditional uses of G. lucidum, implying G. lucidum-derived compounds' potential in insulin-resistant diseases and cancer, and might inspire a consideration of Ganoderma meroterpenoids as dietary supplements.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.