Sesquiterpenoids from Inula britannica and Their Potential Effects against Triple-Negative Breast Cancer Cells

Flowers of Inula britannica commercially serve as pharmaceutical herbs in the manufacturing of medicinal products. In the current study, sesquiterpenoids of I. britannica flowers’ extract and their potential effects against triple-negative breast cancer (TNBC) cells were investigated. Eight structurally diverse sesquiterpenoids, including one sesquiterpenoid dimer (1) and seven sesquiterpenoid monomers (2–8) were isolated from this source. The structures of all compounds were elucidated by 1D/2D NMR data, and their absolute configurations were discerned by single crystal X-ray diffraction. All of the compounds were tested for their potential effects against TNBC. Specifically, 5 displayed strong antiproliferative potency against TNBC cells with a high selective index (SI) on MCF-7 cells (SI > 4 of IC50 on MDA-MB-468/IC50 on MCF-7), and dimer 1 (IC50 = 8.82 ± 0.85 μM) showed better antiproliferative potency against MCF-7 cells than the other monomers did (2–8) (IC50 > 20 μM). To our best knowledge, compound 5 is the first sesquiterpenoid targeting TNBC cells.


Introduction
The biggest cancer-related disease for women worldwide remains breast cancer (BrCa) [1,2]. BrCa subtypes are defined by their histopathological appearance and expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2). Most of the cases are ER-positive and PR-positive BrCa, and hormone therapy normally shows really good effects in them [3]. HER2-positive BrCa takes about 15-20% of all kinds of BrCa. Today, numerous targeted anti-HER2 medications have been authorized for use in treating HER2-positive BrCa [4]. Unfortunately, 10-20% of BrCa is known as triple-negative breast cancer (TNBC) and shows a negative result for ER, PR and HER2 expression. TNBC, with poor overall survival (OS) and an aggressive clinical course, is a poorly understood BrCa type. The incidence of TNBC is amazingly high among overweight [5], non-Hispanic black, and younger women [3,5,6].
Because of the antioxidant and anti-neuroinflammatory activities they have, naturalproduct-derived bioactive agents have attracted attention on the development of preventive neuroprotectants or nutraceuticals for the treatment of neurodegenerative disorders [7][8][9][10]. In Eastern Asia, the Inula britannica plant has been used to treat disorders of the digestive system, bronchitis and inflammation for many years [11,12]. Numerous biologically active sesquiterpene lactones have been identified from this plant, according to earlier phytochemical studies [13]. Several studies have shown that certain sesquiterpene lactones can prevent breast cancer cells from migrating and invading [14,15]. For example, eupatolide lowers inflammation by inhibiting the nuclear factor-light-chain-enhancer of activated B cells, as well as preventing the production of tumor necrosis factor (TNF) and NO [16][17][18]. Moreover, it is also reported that eupatolide could play an essential role in the sensitization of TRAIL-induced apoptosis in human breast cancer cells. Therefore, it is Molecules 2022, 27, 5230 2 of 7 particularly important to explore novel bioactive metabolites from I. britannica flowers and their anti-tumor activities [13].
NO [16][17][18]. Moreover, it is also reported that eupatolide could play an essential role in the sensitization of TRAIL-induced apoptosis in human breast cancer cells. Therefore, i is particularly important to explore novel bioactive metabolites from I. britannica flowers and their anti-tumor activities [13].

Plant Materials
The I. britannica flowers were collected in October 2017 from the river-bed region of Qinling Moutain in Baoji City of Shaanxi province, China. Maintained dry and ventilated, the flowers were identified by Prof. Jun-Mian Tian. A voucher specimen (TJJ-201700119) was kept in a cool and dry environment of Shaanxi Key Laboratory of Natural Products & Chemical Biology, Northwest A&F University.

X-ray Crystallographic Analysis of 4
Crystal Data for C 15

Cell Culture
MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells were seeded in T75 flasks at 2 × 10 6 cells/flask in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), a 1% penicillin/streptomycin mix and were incubated at 37 • C in an atmosphere of 5% CO 2 . The medium was renewed twice a week, and cells were passaged once a week at a subcultivation ratio of 1:3.

SRB Assay
This SRB method was based on SRB's ability to stoichiometrically bind to proteins under mildly acidic conditions before being extracted under basic conditions. As a result, the amount of bound dye could approximately represent the cell mass, from which we could extrapolate to measure cell proliferation [28]. MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells with the densities of 1.2 × 10 4 , 8 × 10 3 , and 8 × 10 3 (cells/well) were inoculated into 96-well plates for 24 h, then, the medium was replaced with a fresh one containing the specified compound. Eight compounds were configured into different media, and each compound was set with five different concentrations of 0.1, 0.5, 1, 5, 10, 20 µM. After 48 h of incubation, cell monolayers were fixed with 10% (w/v) trichloroacetic acid for 12 h, washed with distilled water six times, and stained by SRB (0.4%, w/v) for 30 min. Then, using 1% (v/v) acetic acid, the excess dye was removed from the cells by washing them repeatedly six times. The protein-bound dye was dissolved in 100 µL Tris base solution for optical density (OD) determination at 560 nm using a microplate reader.

Statistical Analysis
All tests were performed at least in triplicate. The data are displayed as the mean ± standard deviation (SD). The exhibited data were investigated by one-way analysis of variance (ANOVA) using Graph prism 8.0.