Inula helenium L. was obtained from the Bozhou traditional Chinese medicine market (Anhui, China). The dried rhizome and root of Inula helenium L. (10.0 kg) were crushed into coarse powder. The powder was soaked with 95% ethanol for 48 h, and extracted with 20-fold 95% ethanol at room temperature by percolation. The combined extract was concentrated in a vacuum to give a crude extract. The crude extract was suspended in 1.5 l warm water and partitioned with ethyl acetate (3×1.5 l). Subsequent to evaporation in reduced pressure, 370.16 g EEIHL was yielded (yield, 3.7%). To verify that EEIHL contained bioactive compounds, a vanillin-sulfuric acid colorimetry assay was applied to establish sesquiterpene lactones in EEIHL (14).

EEIHL was run through an Acquity UPLC system with a BEH C18 column (25°C, 2.1×100 mm, 1.7 µm; Waters Corporation, Milford, MA, USA). CH₃CN and H₂O were used as the mobile phase, and a gradient program was applied as follows: 0–2.5 min, 30% of CH₃CN; 2.5–4 min, 30–45% of CH₃CN; 4–7 min, 45% of CH₃CN; 7–8 min, 45–60% of CH₃CN; 8–9 min, 60% of CH₃CN; 9–10 min, 60–85% of CH₃CN; 10–12 min, 85% of CH₃CN; 12–13 min, 85–30% of CH₃CN; and maintained at 30% of CH₃CN for the next 3 min. The sample solution was 5 mg of EEIHL dissolved in 1.0 ml MeOH, and the injection volume was 1 µl with the flow rate of 0.3 ml/min. The detection wavelength was set at 210 nm.

The presence of alantolactone, isoalantolactone and alloalantolactone was verified by ¹H-NMR. A total of 10 mg of sample was dissolved in CDCl₃, and then detected by NMR. Tetramethylsilane was used as internal standard. The ¹H-NMR was performed with a Bruker Ultra Shield Plus 600 MHz spectrometer (Bruker Corporation, Billerica, MA, USA).

RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Cycletest Plus DNA Reagent kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). DAPI was purchased from Beyotime Biotechnology (Shanghai, China). The Annexin V-FITC Apoptosis kit was purchased from BestBio Company (Shanghai, China). A Mitochondrial Membrane Potential Assay kit was purchased from Signalway Antibody LLC (College Park, MD, USA).

The primary antibodies against cyclin-dependent kinase (CDK)2 (cat. no. ab32147), phosphorylated (p)-CDK2 (cat. no. ab76146), CDK6 (cat. no. ab124821), cyclin D1 (cat. no. ab134175), p-Rb (cat. no. ab184796), cyclin E1 (cat. no. ab135380), Bcl-2 (cat. no. ab32124), Bax (cat. no. ab32503), Bim (cat. no. ab32158), Mcl-1 (cat. no. ab32087), poly(ADP-ribose) polymerase (PARP; cat. no. ab191217), caspase-3 (cat. no. ab13847), X-linked inhibitor of apoptosis protein (XIAP; cat. no. ab21278), AKT (cat. no. ab8805), p-AKT (cat. no. ab38449), signal transducer and activator of transcription 3 (STAT3; cat. no. ab68153), p-STAT3 (cat. no. ab76315) and β-actin (cat. no. ab8226) were purchased from Abcam (Cambridge, MA, USA). Primary antibodies were diluted at 1:1,000.

CFPAC-1 human PDAC cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured with RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37°C in a 5% CO₂ humidified atmosphere. EEIHL was dissolved in DMSO at 100 mg/ml.

The rate of cell proliferation was measured by a Cell Counting Kit-8 (CCK-8) assay (BestBio Company). Cells were cultured in 96-well plates at a concentration of 7×10³/well. Cells were cultured for 24 h and treated with the indicated concentrations of EEIHL. At 48 h of treatment, the supernatant was removed, 100 µl of CCK-8 solution was added to each well and the plates were incubated for a further 2 h at 37°C. Cell viability was quantified by a Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific, Inc.) by the optical density (OD) at 450 nm. Cell viability was calculated as [(OD₄₅₀ of treated cells/OD₄₅₀ of control cells) ×100%]. Three independent experiments were performed.

CFPAC-1 cells were seeded into 6-well plates at a density of 1×10³/well and incubated for 24 h. The cells were then treated with the indicated concentrations of EEIHL. Following 24 h of treatment, the supernatant was removed and cells were cultured for a further two weeks. Then the cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa solution for 15 min at room temperature. Visible colonies were imaged with a ChemiDoc XPS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Exponentially growing cells were seeded in 6-well plates (2×10⁵/well) and cultured overnight in a 5% CO₂ atmosphere at 37°C. Following treatment with EEIHL, CFPAC-1 cells were harvested and washed twice with cold PBS and then fixed in 70% cold ethanol at 4°C overnight. Cells were stained with the Cycletest Plus DNA Reagent kit (BD Biosciences), according to the manufacturer's protocol. Cell cycle distribution was analyzed using a flow cytometer (BD Biosciences). Data were collected by BD FACSDiva software version 8.0.1 (BD Biosciences) and analyzed by ModFit LT for Windows version 4.1.7 (Verity Software House, Topsham, ME, USA). Three independent experiments were performed.

Exponentially growing cells were seeded in 6-well plates (2×10⁵/well) and cultured overnight in a 5% CO₂ atmosphere at 37°C. Following treatment with EEIHL for 48 h, the cells were harvested and washed with PBS. Then the cells were stained with the Annexin V-FITC Apoptosis kit, according to the manufacturer's protocol, and analyzed with a flow cytometer (BD Biosciences). Three independent experiments were performed.

CFPAC-1 cells (8×10⁴ cells/well) were cultured in 24-well plates. Following exposure to EEIHL, cells were fixed with 4% paraformaldehyde for 20 min, and stained with DAPI for 15 min at room temperature. Following washing with PBS, cells were observed under a fluorescence microscope (Ti-E; Nikon Corporation, Tokyo, Japan).

Mitochondrial membrane potential was visualized by the 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethyl-imidacarbocyanine iodide (JC-1) stain from the Mitochondrial Membrane Potential Assay kit. Cells were seeded into 6-well plates at a density of 2×10⁵/well, and cultured for 24 h. Following treatment, the cells were collected, washed with PBS, and incubated with JC-1 for 15 min at 37°C. Subsequent to washing, cells were immediately analyzed using a flow cytometer (BD Biosciences). Three independent experiments were performed.

CFPAC-1 cells (2×10⁵ cells/well, 6-well plate) were incubated with EEIHL for 48 h. Cells were washed with PBS and lysed in cell lysis buffer included in the Caspase-3 assay kit. Caspase-3 activity in cell lysates were determined colorimetrically using a BioVision colorimetric caspase assay kit (BioVision, Inc., Milpitas, CA, USA). Chromophore conjugated peptides, including DEVD-p-nitroanilide (pNA) and VEID-pNA, were substrates for caspase-3, releasing pNA on caspase activity, which was quantified according to the manufacturer's protocol.

Following treatment with different concentrations of EEIHL, total proteins (40 µg) were extracted using radioimmunoprecipitation assay lysing buffer (RIPA; cat. no. P0013B; Beyotime Institute of Biotechnology, Shanghai, China) and subjected to 12% SDS-PAGE prior to being transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% non-fat milk at room temperature for 1 h, and then incubated with specific primary antibodies overnight at 4°C. Subsequent to washing in TBST, membranes were incubated with the secondary antibodies (dilution 1:5,000; HRP Goat Anti-Mouse cat. no. A21010 and HRP Goat Anti-Rabbit cat. no. A21020; Abbkine, Wuhan, Hubei, China) at room temperature for a further 1 h. The protein bands were visualized using an ECL system WBKLS0050 (EMD Millipore, Billerica, MA, USA) and analyzed using Bio-Rad Laboratories Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Exponentially growing cells were seeded in 6-well plates (2×10⁵/well) and cultured overnight in a 5% CO₂ atmosphere at 37°C. Following a 24-h incubation, a 1-ml pipette tip was used to gently scratch the monolayer at the center of the well. Detached cells were washed away with PBS, and the remaining cells were cultured with serum-free medium and the indicated doses of treatment. Cells were incubated for 24 h prior to the capture of images (DS-Ri2; Nikon Corporation).

Total RNA was extracted from cells with TRIzol (Life Technologies; Thermo Fisher Scientific, Inc.), precipitated with isopropyl alcohol, and rinsed with 70% ethanol. Single-strand cDNA was prepared from the purified RNA using PrimeScript RT Master Mix (cat. no. RR036A; Takara Biotechnology Co., Ltd., Dalian, Liaoning, China), followed by qPCR with SYBR-Green (Qiagen GmbH, Hilden, Germany) in the 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling parameters were 94°C for 30 sec followed by 40 cycles of 58°C for 30 sec and 72°C for 30 sec, with one final cycle of 72°C for 90 sec. Three independent experiments were performed. The relative expression of target genes against the reference gene was obtained from 2^−ΔΔCq values (15).

The primers used were: E-Cadherin, forward, 5′-TTCTGCTGCTCTTGCTGTTT-3′, reverse, 5′-TGGCTCAAGTCAAAGTCCTG-3′; Snail, forward, 5′-GAAAGGCCTTCAACTGCAAA-3′, reverse, 5′-TGACATCTGAGTGGGTCTGG-3′; GAPDH, forward, 5′-GAGTCAACGGATTTGGTCGT-3′, reverse, 5′-TTGATTTTGGAGGGATCTCG-3′.

The results are expressed as the mean ± standard deviation of at least three independent experiments. A two-sided Student's t test or one-way analysis of variance followed by a post hoc Fisher's least significant difference test was used to analyze the differences among groups. Graphs were prepared using SigmaPlot software version 12.0 (Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To extract sesquiterpene lactones from crude IHL roots efficiently, ethanol and ethyl acetate were used to partition IHL and produce the final product, EEIHL. A vanillin-sulfuric acid colorimetry assay was then applied to establish sesquiterpene lactones in EEIHL. The color of the mixture was purple or purple-red, indicating the existence of sesquiterpene lactones in EEIHL, as previously described (16). These three compounds were further confirmed by analysis with HPLC (Fig. 1). In addition, ¹H-NMR spectra were used to identify the presence and structure of alantolactone, isoalantolactone and alloalantolactone (Fig. 2).

The anti-proliferative activity of EEIHL against CFPAC-1 PDAC cells was determined by CCK-8 assay and colony formation assay. As shown in Fig. 3A, EEIHL and isoalantolactone dose-dependently inhibited the proliferation of CFPAC-1 cells at 48 h treatment. The inhibition rate was >90% following treatment with 6 µg/ml of EEIHL for 48 h, and the half maximal inhibitory concentration (IC₅₀) was 4.3 and 2.8 µg/ml for EEIHL and isoalantolactone, respectively. In addition, a relatively low concentration of EEIHL inhibited the formation of CFPAC-1 colonies (Fig. 3B). When cells were treated with 2 µg/ml of EEIHL for 24 h and cultured for a further 2 weeks, the number of CFPAC-1 colonies was diminished. At concentrations of 1 or 2 µg/ml, EEIHL demonstrated anti-proliferation activity after 24 h treatment. Therefore, EEIHL potently inhibited the proliferation and colony formation of CFPAC-1 cells.

Following treatment with EEIHL for 24 h, CFPAC-1 cells were stained with propidium iodide (PI) followed by cell cycle analysis. As shown in Fig. 4A and B, 2 µg/ml of EEIHL treatment resulted in a 15% increase in the proportion of cells in the G₀/G₁ phase. The protein expression of total CDK2 and p-CDK2 gradually decreased following treatment with EEIHL (Fig. 4C), whereas the protein level of cyclin D1 or cyclin E1 remained unchanged. Notably, pRb was downregulated following EEIHL treatment, indicating that EEIHL may downregulate CDK2 by affecting pRb.

As the EEIHL concentration was increased, CFPAC-1 cells displayed an increasing extent of mitochondrial-dependent apoptosis. As shown in Fig. 5A and B, 4 and 6 µg/ml of EEIHL treatment induced 27 and 48% apoptosis, respectively, while untreated cells displayed an apoptosis rate of 6%. Meanwhile, 4 µg/ml of EEIHL resulted in shrunken nuclei and intensified DAPI fluorescence (Fig. 5C).

To explore the role of the mitochondria in EEIHL-induced apoptosis, the JC-1 stain was applied to determine the mitochondrial membrane potential (Fig. 6). Following treatment with increasing concentrations of EEIHL, depolarized mitochondrial membrane potential gradually increased to 10.8% (Fig. 6A and B). Proteins determining mitochondrial fate were then analyzed by western blot (Fig. 6D). In accord with the depolarized mitochondrial membrane potential, pro-apoptotic protein Bim was evidently elevated, whereas anti-apoptotic proteins Bcl-2 and Mcl-1 were downregulated, suggesting the crucial role of the mitochondria in EEIHL induced apoptosis. Furthermore, as indicated by the appearance of cleaved PARP and decreased levels of XIAP, EEIHL induced caspase-dependent apoptosis; this was verified by determining the caspase activity following EEIHL treatment (Fig. 6C). In addition, EEIHL induced the decreased expression of p-AKT and p-STAT3, suggesting alternative pathways involved in the anti-proliferation effect of EEIHL (Fig. 6D).

Cell migration was measured using a wound healing assay. When treated with 2, 4 or 6 µg/ml of EEIHL, the migration ability of CFPAC-1 cells was gradually attenuated (Fig. 7A and B). In comparison with isoalantolactone, 4 µg/ml of EEIHL was significantly more potent in impeding CFPAC-1 cell migration. Snail is a transcriptional factor that promotes the repression of E-cadherin, which serves a key role in cell adhesion; the dysregulated transcription of Snail and E-cadherin is associated with the epithelial-mesenchymal transition (EMT) and tumor metastasis. As shown in Fig. 7C, the mRNA level of E-cadherin increased following EEIHL treatment to a greater extent than treatment with isoalantolactone, indicating the inhibitory effect of EEIHL on cell mobility. Meanwhile, the mRNA level of Snail was significantly downregulated, suggesting that EEIHL may also inhibit the Snail-induced regulation of EMT.