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A New Sterol From Sporoderm-Broken Ganoderma sinense Spores and Its Anticancer Activity

A new sterol, ganodermaside E (1), and 4 known sterols, (22E,24R)-3β,5α-dihydroxyergosta-7,22-dien-6-one (2), (22E,24R)-3β,5α,9α-trihydroxyergosta-7,22-diene-6-one (3), (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β-triol (4), and (22E,24R)-ergosta-7,22-diene-3β,5α,6α-triol (5), were isolated for the first time from the sporoderm-broken spores of Ganoderm sinense Zhao, Xu et Zhang. Their structures were determined by spectroscopic techniques such as nuclear magnetic resonance spectroscopy and mass spectrometry. Furthermore, all the compounds were evaluated for their in vitro cytotoxicity and migration inhibition on human non-small-lung cancer A549 cells. Compound 1 exhibited cytotoxicity with a half-maximal inhibitory concentration value of 21.12 ± 1.46 µM. Compound 5 exhibited the strongest and most significant antimetastatic activity at concentrations of 100 and 200 µM.

Besides uncontrolled multiplication, metastasis of the cancer cells also plays an important role in tumor development. In view of the in vivo nutritional deficiency of the cancer cells, herein, an in vitro tumor metastasis model was established by serum starvation stimulation. Table 1 shows that 2, 4, and 5 significantly suppressed the migration of A549 cells induced by serum starvation at a concentration of 200 µM. Among these, only 5 could notably suppress the migration at a concentration of 100 µM. Considering that 1 exhibited significant cytotoxic activity (90.33% ± 4.26%) at a concentration of 30 µM, the concentrations of 1 for the transwell assay were adjusted to 5, 10, and 20 µM. However, 1 showed no significant inhibition for the migration of A549 cells at the 3 concentrations.
Compounds 1 to 5 were isolated for the first time, to the best of our knowledge, from G. sinense. The pharmacological assays showed that compounds 1 and 5 could notably inhibit the proliferation and metastasis, respectively, of A549, suggesting their potential as antineoplastic agents. In vivo studies and further investigations on the specific molecular mechanisms will be carried out to confirm the antiproliferative and antimetastatic effects of the isolated compounds on A549 cells.

Fungal Materials
The spores of G. sinense were collected on August 2018 from the Ganoderma base in an alpine forest of Fujian Province, south-eastern China. This region was developed by the Food Engineering Research Center of the State Ministry of Education, Sun Yat-Sen University. The spores were stored in a dry storage within a narrow temperature range between 5 and 10°C. They were identified by Dr Wenhua He, Institute of Microbiology, Chinese Academy of Sciences. A voucher specimen (HMAS 77207M) has been deposited in the same institution.

Extraction and Isolation
The spores of G. sinense (13.2 kg) were subjected to sporodermbreaking treatment and then extracted by supercritical CO 2 fluid extraction to give a crude extract, as described previously. 6 Data are reported as the mean ± standard error of the mean of at least 3 independent experiments. a P < 0.01, compared to the blank control group. b Cisplatin was used as a positive control. c P < 0.01, compared to the model group. d P < 0.05, compared to the model group.

Transwell Assay
The transwell assay was performed in a 24-well Boyden chamber with an 8 mm pore size polycarbonate membrane (Corning, NY, USA). The A549 cells were treated with compounds 1 to 5 at 3 concentrations in DMEM with 10% FBS for 24 hours and then cultured without serum for another 24 hours. Thereafter, the cells (5 × 10 4 ) were harvested, resuspended in 100 µL DMEM without serum, and injected to the upper chamber. Meanwhile, the lower chamber was infused with 500 µL DMEM and 10% FBS to encourage migration. After 48 hours, the cells that migrated to the surface of the lower chamber were fixed with MeOH, stained with crystal violet, and counted under an optical microscope with 200× magnification. Throughout the transwell assay, the blank control group was the cells cultured with serum, while the model group was the cells treated only with serum starvation. Cisplatin was used as a positive control. The positive control group was the cells cultured with cisplatin and serum starvation. The differences were found to be significant for P < 0.05, 0.01.