A New Human Cancer Cell Proliferation Inhibition Sesquiterpene, Dryofraterpene A, from Medicinal Plant Dryopteris fragrans (L.) Schott

The global burden of cancer continues to increase largely with the aging and growth of the world population. The purpose of the present work was to find new anticancer molecules from a natural source. We utilized chromatographic methods to isolate compounds from medicinal plant Dryopteris fragrans (L.) Schott. The structure of the new compounds was determined by spectroscopic and spectrometric data (1D NMR, 2D NMR, and EMI-MS). Their anti-proliferation effects against five human cancer cell lines including A549, MCF7, HepG2, HeLa, and PC-3 were evaluated by CCK-8 andlactate dehydrogenase (LDH) assay. A new sesquiterpene, (7S, 10S)-2,3-dihydroxy-calamenene-15-carboxylic acid methyl ester (1), and two known compounds (2 and 3) were isolated. The new sesquiterpene was named dryofraterpene A and significantly inhibited cancer cell proliferation without any obvious necrosis below a 10 μM concentration. In conclusion, a novel anticancer sesquiterpene together with two known compounds was isolated, which might be a promising lead compound for the treatment of cancer.


Introduction
Cancer is a major public health problem in a great many parts of the world. In the United States alone, a total of 577,190, 580,350, 585,720, 589,430, and 595,690 deaths from cancer were respectively predicted to occur in 2012-2016 [1][2][3][4][5], and these numbers have been increasing year by year. Great therapy attention has been paid to the development of novel anticancer molecules from natural sources. However, the available chemotherapeutics is often limited due to undesirable drug resistance and side effects [6,7]. It is urgent that new targets for the treatment of cancer are identified.
Dryopteris fragrans (L.) Schott (Chinese name: Xiang-Lin-Mao-Jue) (Figure 1), a deciduous perennial herb from the family Dryopteridaceae, is widely distributed in Asia-temperate, Europe, and North America [8]. In the north of China, it has drawn wide attention due to its folk effect on various dermatosis and rheumatoid arthritis [9]. Many constituents of D. fragrans have exhibited various biological activities, such as anticancer [10], anti-inflammatory [11], antibacterial [12], antifungal [13], and antioxidant activities [14]. Therefore, with the intent of discovering new compounds with potential antitumor properties, we performed two fractionations of the EtOAc extract and obtained a new sesquiterpene (1) together with two known compounds (2 and 3) (Figure 2). We are strongly interested in the biological activity of the new sesquiterpene. Human cancer cell proliferation inhibition activity was tested on A549, MCF7, HepG2, HeLa, and PC-3 cells, which are the in vitro models of lung, breast, liver, cervical, and prostate cancer that are the leading cause of cancer death in more or less developed countries [15]. The novel sesquiterpene showed the inhibitory effects of cancer cell growth.

Identification of Isolated Compounds
Compound 1 was colorless crystals, m.p. 115 °C, [α] 25 D −37.9 (c 0.0026, CDCl3). The molecular formula was assigned as C16H22O4 on the basis of the [M + H] + peak at m/z 279.1590 (Calcd for C16H23O4, 279.1596) in its high resolution electrospray ionization mass spectroscopy (HR-ESI-MS), and this could be supported by evidence from 13 C-NMR combined with the distortionless enhancement by polarization transfer (DEPT) spectrum ( Table 1). The infrared radiation (IR) spectrum of compound 1 showed a presence of the hydroxyl group (3431 cm −1 ), an aromatic ring (1629 cm −1 ), and an ester carbonyl group (1721 cm −1 ).  Therefore, with the intent of discovering new compounds with potential antitumor properties, we performed two fractionations of the EtOAc extract and obtained a new sesquiterpene (1) together with two known compounds (2 and 3) (Figure 2). We are strongly interested in the biological activity of the new sesquiterpene. Human cancer cell proliferation inhibition activity was tested on A549, MCF7, HepG2, HeLa, and PC-3 cells, which are the in vitro models of lung, breast, liver, cervical, and prostate cancer that are the leading cause of cancer death in more or less developed countries [15]. The novel sesquiterpene showed the inhibitory effects of cancer cell growth. Therefore, with the intent of discovering new compounds with potential antitumor properties, we performed two fractionations of the EtOAc extract and obtained a new sesquiterpene (1) together with two known compounds (2 and 3) ( Figure 2). We are strongly interested in the biological activity of the new sesquiterpene. Human cancer cell proliferation inhibition activity was tested on A549, MCF7, HepG2, HeLa, and PC-3 cells, which are the in vitro models of lung, breast, liver, cervical, and prostate cancer that are the leading cause of cancer death in more or less developed countries [15]. The novel sesquiterpene showed the inhibitory effects of cancer cell growth.

Effects of Compounds on Cancer Cell Proliferation
By the CCK-8 assay, dryofraterpene A (1) was evaluated for cancer cell proliferation inhibition activities in vitro against A549 (lung cancer), MCF7 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer), and PC-3 (prostate cancer) human cell lines, using taxol as a positive control. As summarized in Table 2, dryofraterpene A (1) significantly inhibited the growth of all the five cell lines. At the same time, we observed a decrease in the total cell number and an increase in floating cells, with cell shrinkage and cytoplasm vacuolization in dryofraterpene A-treated cancer cells by the inverted phase-contrast microscope (data not shown).  The 2D-nuclear overhauser effect spectroscopy (NOESY) spectrum established the relative configuration of the stereocenters for compound 1. A correlation between Me-16 and Me-14 confirmed a cis-calamenene, according to reported literature [16,17]. Based on the above spectroscopic analysis, the structure of compound 1 was determined to be (7S, 10S)-2,3-dihydroxy-calamenene-15-carboxylic acid methyl ester and was named as dryofraterpene A.

Effects of Compounds on Cancer Cell Proliferation
By the CCK-8 assay, dryofraterpene A (1) was evaluated for cancer cell proliferation inhibition activities in vitro against A549 (lung cancer), MCF7 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer), and PC-3 (prostate cancer) human cell lines, using taxol as a positive control. As summarized in Table 2, dryofraterpene A (1) significantly inhibited the growth of all the five cell lines. At the same time, we observed a decrease in the total cell number and an increase in floating cells, with cell shrinkage and cytoplasm vacuolization in dryofraterpene A-treated cancer cells by the inverted phase-contrast microscope (data not shown). * Results are expressed as IC 50 values in µM, which represent the mean ± standard error (SE) of three independent assays. ** Taxol was as positive control.
The LDH assay detects the amount of LDH released by cells with damaged membranes as indicator of necrosis. Forty-eight-hour treatment with dryofraterpene A (1) did not affect the concentration of LDH in the supernatant of culture medium of five cancer cell lines (99.9% ± 8.7%, 103.2% ± 7.0%, 98.2% ± 6.4%, 100.5% ± 4.3%, and 101.6% ± 7.4% at 10 µM, respectively, p > 0.05). This suggests an anti-proliferative effect of dryofraterpene A (1) on cancer cells without any obvious necrosis, perhaps with inducing apoptosis, below the dose of at least 10 µM, which might be used for treatment in future experiments [20].

Cell Culture
Human A549, MCF7, HepG2, HeLa and PC-3 cells were obtained from Cell Library of Committee on Type Culture Collection of Chinese Academy of Sciences. Cultures were maintained in 95% air and 5% CO 2 at 37 • C in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin.

LDH Assay
Leakage of LDH to the cell culture medium indicates cell membrane damage. LDH assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). After cells were exposed to dryofraterpene A (1) (0 and 10 µM) for 48 h, each culture medium was centrifuged at 250 g for 10 min. Supernatant was transferred to a 96-well culture plate to determine the amount of LDH according to the manual of the LDH assay kit. LDH activity is reported as a percentage relative to control level [25]. Absorbance of samples was measured at 450 nm. Data were obtained from three independent assays.