Antiausterity Activity of Secondary Metabolites from the Roots of Ferula hezarlalehzarica against the PANC-1 Human Pancreatic Cancer Cell Line

Human pancreatic cancer is one of the most aggressive types of cancer, with a high mortality rate. Due to the high tolerance of such cancer cells to nutrient starvation conditions, they can survive in a hypovascular tumor microenvironment. In this study, the dichloromethane extract of the roots of Ferula hezarlalehzarica showed potent preferential cytotoxic activity with a PC50 value of 0.78 μg/mL. Phytochemical investigation of this extract led to the isolation of 18 compounds, including one new sesquiterpenoid (6) and one new monoterpenoid (18). All isolated compounds were evaluated for their preferential cytotoxicity against PANC-1 human pancreatic cancer cells by employing an antiausterity strategy. Among them, ferutinin (2) was identified as the most active compound, with a PC50 value of 0.72 μM. In addition, the real-time effect of ferutinin (2) and compound 6 against PANC-1 cells, exposed to a nutrient-deprived medium (NDM), showed cell shrinkage, leading to cancer cell death within a short period of exposure. Compounds 2 and 6 also inhibited colony formation of PANC-1 cells. The present study indicates that the dichloromethane extract of the roots of F. hezarlalehzarica is a rich source of bioactive compounds for targeting PANC-1 cells.

H uman pancreatic cancer is one of the most aggressive and lethal types of cancer, with median survival rates of less than six months after diagnosis. 1 It frequently occurs in industrialized countries, and reports show that it is the seventh leading cause of cancer-related mortality worldwide, with a 5year survival rate of only 9%. 2 Inefficient diagnosis and the remarkable drug resistance of pancreatic cancer cells have elevated the demand for developing new therapies. Recent studies have shown that the microenvironment of PANC-1 cells and the activation of stellate cells lead to dense stroma formation, providing an immunosuppressive environment and resistance to chemotherapy that accounts for the survival of these cells. 3 Tumor cells, therefore, grow rapidly and invasively. An antiausterity strategy is a method of targeting resistance of pancreatic cancer cells to nutrient starvation conditions. 4 This method was developed previously and applied successfully for the discovery of potent natural products such as arctigenin, formulated as an enriched extract (GBS-01), which has completed phase IIa clinical trials for targeting pancreatic cancer in gemcitabine refractory pancreatic cancer patients. 5 In the framework of a recent screening program on the antiausterity activity of more than 66 plants from Iran, Ferula hezarlalehzarica Ajani (Apiaceae) showed promising activity on PANC-1 cells with a PC 50 value of 0.78 μg/mL. The genus Ferula comprises more than 170 species worldwide, of which 30 grow in Iran, including 16 species endemic to the country. 6 These plants have been used traditionally for treating skin infections, hysteria, and stomach disorders. 6 F. hezarlalehzarica was identified for the first time in 2008, 7 and there have been no prior phytochemical investigations of this plant to date. In the present study, reported for the first time are the isolation, identification, and determination of the absolute configuration of secondary metabolites of F. hezarlalehzarica. Furthermore, the antiausterity activity of isolated compounds was evaluated, and the effects of two of the most active compounds, 2 and 6, were investigated on cell morphology and colony formation of PANC-1 cells.

■ RESULTS AND DISCUSSION
Methanolic extracts of 66 plants from Iran were screened for their antiausterity activity on human pancreatic cancer cells (PANC-1) in nutrient starvation conditions using a previously established method. 4 The root extract of F. hezarlalehzarica showed the most promising activity, with a PC 50 value of 0.78 μg/mL ( Figure S1-A, Supporting Information). HPLC-DAD analysis of the extract indicated that the major compounds were rather lipophilic. Thus, extraction of the root material was performed with dichloromethane, followed by a reassessment of its activity ( Figure S1-B, Supporting Information). Subsequent purification steps of the dichloromethane extract led to the isolation of a new daucane-type sesquiterpenoid (6) and a new monoterpenoid (18), along with 16 known monoand sesquiterpene derivatives ( Figure 1). Jaeschkeanadiol (1), 8 ferutinin (2), 9 teferin (3), 9 10α-hydroxyferutinin (4), 10 kuhistanicaol G (5), 11 felikiol (7), 12 felikiol 8-angelate (8), 12 two lancenotriol p-hydroxybenzoate isomers (9S = α-OH; 9R = β-OH) (9, 10), 13 lancerodiol p-hydroxybenzoate (11), 13 webiol 3-angelate (12), 12 8,9-epoxyferutinin (13), 14 (±)-tschimgin (14), 15,16 (±)-tschimganin (15), 15,16 fenoferin (16), 17 and stylosin (17) 17 were isolated previously from different Ferula species in the past. Although their relative configurations were established, the absolute configuration of most of them was not determined except for compound 1, but with somewhat confusing literature values. 9−14 For this purpose, electronic circular dichroism spectra of selected compounds were simulated for the first time in the present study by quantum chemical calculation methods and compared with experimental spectra. Compounds 2, 4, 5, 9−11, and 13 share a p-hydroxybenzoic acid ester moiety or, in the case of 3, the corresponding 3-methoxy-4-hydroxybenzoic acid ester unit, which are suitable as ECD chromophores by giving two Cotton effects around 250 and 225 nm. Since all the abovementioned compounds showed the same relative configuration at C-6 (according to the NOESY experiments performed) and displayed similar experimental ECD spectra, calculations were only performed for compound 13, and the results obtained were extended to compounds 2−5 and 9−11 (Figures S18− S20, Supporting Information). For compounds 6,7,8,12, and 18, ECD calculations were performed individually. As the NOESY correlations were not conclusive in the case of compound 12, NMR chemical shift calculations along with DP4+ probability evaluation were applied for the determination of its relative configuration prior to ECD calculation. In contrast, compounds 14 to 17 were isolated as racemic mixtures, since no optical activity was observed during the measurement of the respective optical rotations. The structures of the compounds isolated with their absolute configuration are shown in Figure 1.
NOESY correlations revealed NOEs from H-5 to H-10 and from H-2 to H-9 and H-7b, resulting in a relative configuration of 1R*, 2S*, 4R*, and 5S*. Subsequently, the 3D structure was subjected to conformational analysis followed by geometrical optimization and ECD simulation, which led to the determination of the absolute configuration of compound 18 as 1R, 2S, 4R, and 5S ( Figure 3).
Preferential Cytotoxicity against PANC-1 Cells. Human PANC-1 cells show a remarkable tolerance to nutrient-deprived conditions and can survive under an extreme austere hypovascular tumor microenvironment 19 for prolonged time periods. An antiausterity strategy targets the tolerance of cancer cells to nutrition starvation, thereby killing the cancer cells selectively. Previous studies have shown the effectiveness of this approach for the discovery of potential anticancer agents. 20,21 All compounds isolated therefore were investigated for their preferential cytotoxic activity in the nutrient-deprived medium (NDM) along with the nutrient-rich medium (DMEM). The activity is expressed as the PC 50 value, which is the concentration at which 50% of cancer cells were preferentially killed in the NDM without apparent toxicity in the DMEM. As shown in Table 2, most of the isolated  Journal of Natural Products pubs.acs.org/jnp Article compounds, in particular compounds 2, 3, and 10, exhibited promising antiausterity activity with PC 50 values of 0.7, 1.2, and 1.3 μM, respectively. The potency of compound 2 was equivalent to that of the positive control, arctigenin (PC 50 = 0.9 μM), an established antiausterity agent. As most of the active compounds bear a benzoic acid ester unit, the activities of these units (p-hydroxybenzoic acid and p-benzoic acid methyl ester) were additionally assessed as pure compounds. Interestingly, no preferential cytotoxic activity was observed at the investigated concentrations, suggesting that a combination of a terpene unit with an ester moiety is crucial for the activity of these compounds, which was supported also by the high PC 50 value of 1 (90.5 μM), representing an unfunctionalized sesquiterpene alcohol. Subsequently, all isolated compounds were also tested in a nutrient-rich medium (DMEM), and, interestingly, no cytotoxicity was observed at the applied concentrations.
In the next step, ferutinin (2) and its new and active analogue 6 were selected for further investigations on PANC-1 cells to determine their effects on cell death in real time. Cells were treated with 5 μM of the compounds selected in NDM and incubated in a stage-top CO 2 incubator at 37°C on a digital cell imaging station, and images were captured every 10 min under the phase-contrast mode for 24 h. As shown in Based on the previous results, the effects of 2 and 6 on colony formation of PANC-1 cells were evaluated. This assay is used for the investigation of the development and chemosensitivity of early tumors. 22 In order to examine the effects of compounds 2 and 6 on colony formation, PANC-1 cells were treated with three different concentrations (12.5, 25, and 50 μM). As shown in Figure 6, compounds 2 and 6 decreased colony formation of PANC-1 cells significantly in a concentration-dependent manner. Treatment of PANC-1 cells with a concentration of 12.5 μM of compound 2 could further inhibit the growth of the cells after 10 days by a factor of 50% when compared to a control. Although compound 2 was the most active in the preferential cytotoxicity assay, its analogue, compound 6, reduced colony formation by 80% at a concentration of 12.5 μM.
Ferutinin (2) was isolated previously from different Ferula species, 11,13,14,18,23 and previous investigations have indicated its cytotoxic activity against different cancer cell lines. 24,25 In a study performed by Arghiani et al., 26 ferutinin (2) revealed a discernible antitumor activity on human colon cancer cells in vivo and was able to suppress tumor growth significantly. Ferutinin (2) and other isolated derivatives are also potential agonists (ERα) or antagonists (ERβ) of estrogen receptor subtypes, 27 among which ferutinin (2) acts as a nonsteroidal phytoestrogen at the ERα subtype with an EC 50 value of 33.1 nM. 28 Among all these studies, there has been no prior work of the potential antineoplastic activities of ferutinin (2) and its derivatives targeting pancreatic cancer cells. In this study, ferutinin (2) and its derivatives exhibited promising antiausterity activity on PANC-1 cells. Further investigation revealed that ferutinin (2) and compound 6 affect cell membrane integrity and inhibited cell colony formation. Therefore, the roots of F. hezarlalehzarica may represent a valuable source of potential antitumor compounds that target pancreatic cancer cells.
Preferential Cytotoxicity Assay against PANC-1 Cells. The plant dichloromethane extract and all isolated compounds (1−18) were evaluated for their preferential cytotoxicity activity against human pancreatic cancer cells (PANC-1), based on a previously established method. 4 Assessment of Morphology and Apoptosis of PANC-1 Cancer Cells. Compounds 2 and 6, as the most active and as a new compound, respectively, were selected to investigate their realtime effect on cell morphology and apoptosis of PANC-1 cells. For this study, PANC-1 cells were seeded in 12-well plates (1 × 10 6 ) and incubated in a humidified CO 2 incubator for 24 h for cell attachment. The cells were then washed twice with PBS and treated with vehicle control or test compounds at a concentration of 5 μM in NDM and subjected to time-lapse microscopy using an EVOS digital microscope fitted with stage-top incubator in an interval of 10 min for 24 h.
Colony Formation Assay. PANC-1 cells were plated in 12-well plates at a density of 500 cells/well in DMEM (1 mL/well) and incubated at 37°C under humidified 5% CO 2 for 24 h for cell attachment. The cells were treated with compound 2 or compound 6 at concentrations of 12.5, 25, and 50 μM in DMEM and allowed to grow for 10 days. After 10 days, cells were washed with PBS, fixed with 4% formaldehyde, and stained with crystal violet for 10 min before measurement. Each experiment was repeated three times. The colony area measurement was carried out by the ImageJ plugin "Colony Area". Statistical differences were analyzed with GraphPad Prism using the Student's t test. p < 0.05 was considered significant (*).
Copies of spectroscopic data for new compounds 6 and 18, antiausterity activity of the extract, experimental and ECD calculation of other known isolated compounds, and DP4+ calculation results of compound 12 (PDF)