2.1 Plant material

The roots of Ferula ferulaeoides (Steud.) Korovin were collected in Shihezi City, Xinjiang Uygur Autonomous Region, China, in May 2019, and identified by Professor Kejian Pang (Shihezi University, Shihezi, China). In the School of Pharmaceutical Sciences at South-Central Minzu University in Wuhan, China, a voucher specimen (No. SC0426) was deposited.

2.2 Preparation and chemical interpretation of FF-EtOAc

Mechanically crushed F. ferulaeoides roots had been air-dried, and 90 g of the powder was used for extracting by speed extraction at 80℃, 10 MPa with petroleum ether, ethyl acetate, and methanol (each solvent, 4 cycles; each cycle 15 min) to yield petroleum ether extract (4.03 g), ethyl acetate extract (FF-EtOAc, 10.27 g) and methanol extract (6.11 g), and the extraction rates were 4.47%, 11.4%, and 6.78%.

The chemical profile of FF-EtOAc was conducted by UPLC-Q-TOF-MS, and it was employed by using an ultra-high performance liquid chromatography (UPLC) system (Waters Corp, Manchester, England) consisting of a binary pump, a degasser, an auto-sampler, and a column compartment with a high throughput G2Si High-definition mass spectrometer (Waters Q-TOF SYNAPTTM, Waters Corp, Manchester, England), and equipped with an electrospray (ESI) interface. A Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) was used for the chemical profiling of FF-EtOAc. The 0.1% aqueous formic acid (A) and acetonitrile (B) made up the mobile phase. This system was used for chromatographic separation at 25 °C. Gradient elution with a flow rate of 0.4 mL/min was performed as follows: 2% − 95% B at 0––25 min. Leucine enkephalin was employed as the precise mass calibration solution for the MS analysis, which was conducted using an electrospray ionization (ESI) source in both positive and negative ion modes. 350 °C was the temperature of the desolvation gas. Cone and desolvation gas flow rates were established at 50 L/h and 600 L/h, respectively. In the positive and negative ion modes, the capillary, cone, and extraction cone voltages were set to 3.0 and 2.5 V, respectively. With a low collision energy of 6 V and a high collision energy of 20–80 V, MSE was used for MS/MS analysis. The scan area was set at m/z 50–1200.

2.3 Chemicals and reagents

The MTT chemicals were from neoFroxx (Einhausen, Germany), and the Annexin V-FITC/PI Double-stained Apoptosis Detection Kit came from BestBio (Shanghai, China). The Hoechst 33258 reagents, BCA protein quantification kit, SDS–PAGE sample loading buffer, and RIPA lysis solution were purchased from Beyotime Biotechnology (Nantong City, Jiangsu Province, China). We purchased the eECL Western blot Kit from Bioprimacy (Wuhan China). Dulbecco's modified eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 were purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) from Newzerum (Christchurch New Zealand) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) were obtained from Hyclone (Logan, UT, USA). Antibodies of MMP-9, Bcl-2, Caspase-3, PARP, Akt, p-Akt (Ser473), Bad, p-Bad (Ser136), p-GSK-3α/β (Ser 21/9), GSK-3α/β and PTEN were obtained from Cell Signaling Technology (Danvers, MA, USA). Caspase-9 and Bax were purchased from ABclonal Biotech (Wuhan, Hubei, China). β-actin was obtained from Bioprimacy (Wuhan China). We purchased the matching secondary antibody from ABclonal Biotech (Wuhan, Hubei, China).

2.4 Cell culture

The human embryonic kidney (HEK) cell lines HEK293 and esophageal cancer (EC) cells (Eca-109 and KYSE150) were purchased from the American Type Culture Collection (Manassas, USA). Eca-109 cells, KYSE-150 cells, and HEK293 cells were grown in a medium containing 90% DMEM or RPMI-1640, 10% FBS, and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin), DMEM is used for Eca-109 cells and HEK-293 cells and RPMI-1640 is used for KESE-150 cells. These cells were cultured in a cell culture incubator (Heal Force, Shanghai, China). The culture conditions were set at 5% CO₂, 37 °C.

2.5 Cell viability test

The noxiousness of FF-EtOAc toward the Eca-109, KYSE150, and HEK293 cell lines was assessed using the MTT test. Eca-109, KYSE150, and HEK293 cells (1 × 10⁵ cells/well) were planted in 96-well plates and cultivated until 90% confluence was reached. Then, 0, 10, 20, 30, 40, and 50 μg/mL FF-EtOAc was applied to Eca-109 and KYSE150 for 6, 12, or 24 h, and HEK293 cells for 12 h. After treating over and discarding the FF-EtOAc, MTT (5 mg/mL) was administered to each well for 30 min in a culture incubator with 37 °C and 5% CO₂. Then, aspirate the MTT solution and add 150 mL of DMSO to each well before using a microplate reader to measure the optical density (OD) value at 570 nm. (Thermo Scientific, CA, USA). Calculating the inhibition rate uses the following formula: inhibition rate (%) = [1 − (OD _sample – OD _blank) / (OD _control – OD _blank)] × 100 (Chen et al., 2019). The concentration of FF-EtOAc at which the cell inhibition rate was 50% as determined by MTT assays and estimated by the GraphPad Prism 8.0 Software (GraphPad Prism Software, Inc.) is known as the IC₅₀ value.

2.6 Histological morphology and Hoechst 33258 staining of cells

EC cells were sowed in 6-well plates (2 × 10⁵ cells/well) and cultivated until 90% confluence was achieved. The EC cells were treated with 0, 10, 20, and 30 μg/mL FF-EtOAc within 12 h. Then using an inverted phase contrast microscope, the cellular morphological alterations were captured at a 20 × magnification (Nikon, ECLIPSE-Ti, Japan). Following treatment, the cells were treated with Hoechst 33258 solution (5 μg /mL) for 30 min in the dark before being fixed for 15 min with a fixing solution (methanol: acetic acid = 3: 1). Then, a fluorescent inverted microscope (Nikon, ECLIPSE-Ti, Japan) was used to observe the cell morphology before a 40 × magnification photograph was taken.

2.7 Cell migration assay

EC cells in the logarithmic phase were seeded on a 6-well plate (1 × 10⁵ /mL /well) for the cell migration experiment. EC cells were treated with 0, 5, 7.5, and 10 μg/mL FF-EtOAc for 12 and 24 h. Using a 200 µL pipette tip, the center of each well was scratched. Scratch images were then captured after 0, 12, and 24 h at a magnification of 20 ×. ImageJ was used to measure and record the scratch width.

For the Transwell migration assay, EC cells were inoculated in Transwell upper compartment (Corning, NY, USA) for 12 h, lower compartment with DEME, or 1640 without FBS. The FF-EtOAc solution was added to the upper compartment and treated with 0, 5, 7.5, and 10 μg/mL FF-EtOAc for 12 h. After drug advertising has ended, absorb and clean the upper compartment and lower compartment. Next, fixed with anhydrous ethanol for 10–20 min and cleaned three times with ultrapure water. The fluorescence inverted microscope was for observing the amount of cell movement and crystalline violet staining. In three randomly selected fields, the number of cells moving through the membrane was counted. The experimental results were analyzed by ImageJ.

2.8 Annexin V-FITC/PI double staining assay and measurement of apoptosis

EC cells were sowed into 6-well plates (2 × 10⁵ cells/well), and cultivated until 70% − 80% confluence was achieved. Within 12 h, the EC cells were treated with 0 and 30 μg/mL FF-EtOAc. The EC cells were digested with trypsin without ethylenediaminetetraacetic acid (EDTA) (Hyclone, Logan, UT, USA) and centrifuge at approximately 300–500 g force, 2–8 °C, 5 min, and the suspended cells were collected. Next, the EC cells were washed four times with cold PBS (Hyclone, Logan, UT, USA) and centrifuged. Then, 400 μL Annexin V Binding Buffer (1 × ) was added to suspend the cells. Add 5 μL of Annexin V-FITC (EGFP/Alexa Fluor488) staining solution to the cell suspension, blend and incubate for 15 min at 2–8 ℃ under light-proof conditions. Add 5–10 μL of PI staining solution and gently mix and incubate for 2–5 min at 2–8 °C under light-proof conditions. After staining and qualitative fluorescence microscopy examination, using flow cytometry, the percentage of apoptotic cells was determined. The percentage of apoptotic cells was examined using FlowJo10 software.

2.9 Protein extraction and Western blot analysis

EC cells were seeded into 6-well plates (2 × 10⁵ cells/well) and cultivated until 70% − 80% confluence was achieved. Then incubated with 0, 10, 20, and 30 μg/mL FF-EtOAc for 24 h, the EC cells were collected by centrifugation and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), phosphatase inhibitor cocktail A (Beyotime, Shanghai, China) and phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride (PMSF) (Boisharp, Hefei, China) to extract the proteins. The protein concentration in the supernatant was measured using the bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China). Then, the protein was stored at −80 °C until required. The same produced for protein extraction from rat tumor tissue was used. Similar sums of the protein were isolated by 10 % or 12.5 % SDS-PAGE gel electrophoresis (Epizyme Biomedical Technology, Shanghai, China) at room temperature and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA) on the ice. The membranes were jammed by 5% Bovine serum albumin (BSA) tris-buffered saline with tween 20 (0.5%) for 2 h. Subsequently, the membranes were incubated with the secondary antibody (1: 10000) at 37 °C for 2 h after being combined with the primary antibody (1: 1000) at 4 °C for overnight. The HRP ECL system (Bio-Rad, CA, USA) is a tool used to visualize protein bands and it is used for our protein bands. The Grayscale values are used for data analysis by Image lab software.

2.10 EC-bearing nude mouse and in vivo treatment

The four weeks old of athymic nude mice were bought from Beijing HFK Biological Science Co., Ltd. (SCXK 2019–0008). The proper institutional, national, and/or international standards for the handling and usage of animals were adhered to. The Experimental Animal Care and Use Committee of South-Central Minzu University's ethical guidelines were followed at all times when using animals in research. After all nude mice were acclimatized for one week. By subcutaneously injecting Eca-109 cells into the mouse's right side of the back, the model of an Eca-109-bearing nude mouse was produced (He et al., 2013, Huang et al., 2020). When the nude mouse’s tumor volume achieved 100 mm³, they were stochastically divided into 3 groups and treated with 0, 60, and 120 mg/kg FF-EtOAc every other day for 21 days. The nude mouse’s tumor volume was measured by a vernier caliper every other day. Tumor volume (V) was calculated as described below: V = 0.5 × width² × length. After 21 days of FF-EtOAc treatment, nude mice were dissected and collected for further analysis of transplanted tumors along with the liver, kidney, and spleen. The Experimental Animal Care and Use Committee of South-Central Minzu University's ethical guidelines were followed in animal experiments and approved (Approval Number: S08921080A).

2.11 TdT-mediated dUTP nick-end labeling (TUNEL assay)

Each specimen of the xenograft mice tissues was cut into slices, fixed, and then embedded in paraffin for TUNEL staining. Following TUNEL analysis, 200 × magnification photos were taken of each tumor fraction.

2.12 Immunohistochemistry (IHC) assay

Each section of the xenograft mice tissues was chosen for immunohistochemistry, and the primary antibody was incubated with it before the corresponding peroxidase-conjugated horseradish secondary antibody was incubated with it and coating for observation. The tumor samples were captured on camera with 200 × magnification.

2.13 Hematoxylin-eosin (HE) staining

Each section of xenograft of nude mice samples was stained hematoxylin and eosin. The tumor samples were captured on camera with 200 × magnification.

2.14 Statistical analysis

Three independent experiments are used to calculate the mean and SD of each set of data. Statistics were analyzed using GraphPad Prism 8.0 Software. ANOVA was used to analyze statistical differences between the FF-EtOAc treated groups, and P values < 0.05 were considered significant (*P < 0.05, **P < 0.01 or ***P < 0.001).

3.1 Chemical composition analysis of FF-EtOAc

The roots of F. ferulaeoides were dried and speed-extracted to produce FF-EtOAc. By searching databases such as China National Knowledge Infrastructure, PubMed, PubChem, and Reaxys, we obtained the complete compound information for F. ferulaeoides. And a self-building database of chemical compounds was established by UNIFI software, including relative molecular mass, molecular formula, CAS number, and mol file of structural formula for each chemical composition. Among them, a total of 42 compounds were listed. High-resolution mass spectrometry data of the main chemical constituents of FF-EtOAc were rapidly acquired by the UPLC-Q-TOF/MS method. The base peak intensity (BPI) chromatograms of FF-EtOAc in the negative and positive ion modes and the basic peak intensity (BPI) chromatograms of FF-EtOAc from in the ultraviolet mode were shown in Fig. 1. We utilized the UNIFI information management platform for swift matching of its mass spectrometry data. Upon closer manual examination, 10 principal chemical constituents were identified in FF-EtOAc, predominantly furanocoumarins. Detailed insights into these constituents can be found in Fig. 1, Fig. 2, and Table 1.

Close

Fig. 1

UPLC-Q-TOF/MS analysis of FF-EtOAc. (A) The basic peak intensity (BPI) chromatogram of FF-EtOAc from UPLC-Q-TOF/MS analysis in the ultraviolet mode at 280 nm. (B) The ESIMS chromatogram of FF-EtOAc in the positive ion mode. (C) The ESIMS chromatogram of FF-EtOAc in the negative ion mode.

Export to PPT

Close

Fig. 2

Structural diagram of the chemical components in FF-EtOAc.

Export to PPT

3.2 Effect of FF-EtOAc on the proliferation and morphology of EC cells

The anti-proliferative effect of FF-EtOAc on EC cells was evaluated by MTT assay, and the IC₅₀ values and cell survival rates of FF-EtOAc were obtained (Fig. 3A and B). FF-EtOAc had lower toxicity to HEK293 cells than the other two types of EC cells (Fig. 3C). Furthermore, observation of cell morphology through an inverted microscope showed that FF-EtOAc induced cellular shrinkage, floating, deformation, and detachment from the cell matrix (Fig. 3D). The results showed that FF-EtOAc markedly suppressed EC cells viability and proliferation in vitro.

Close

Fig. 3

FF-EtOAc inhibited the development of EC cells. (A and B) MTT assays were used to determine Eca-109 and KYSE-150 cells inhibition rates and IC₅₀ values after FF-EtOAc incubation for 6, 12, or 24 h (0, 10, 20, 30, 40, and 50 μg/mL). (C) MTT assays were used to determine Eca-109, KYSE-150 and HEK293 cells inhibition rates and IC₅₀ values after FF-EtOAc incubation 12 h (0, 10, 20, 30, 40, and 50 μg/mL). (D) The morphology of two EC cells after incubation with FF-EtOAc (0, 10, 20, and 30 μg/mL) for 12 h.

Export to PPT

3.3 Effect of FF-EtOAc on the migration of EC cells

Cell migration is the basis for establishing and maintaining the normal organization of multicellular organisms, and restraining the migration of cancer cells is one way to reduce the spread of highly malignant cancer cells (Trepat et al., 2012). The effects of FF-EtOAc on EC cell migration were investigated using both scratch tests and Transwell assays. The expression of Matrix Metalloproteinases is associated with matrix remodeling, particularly cancer invasion, and angiogenesis (Shay et al., 2015). This will be investigated as a mechanism of action, whereby we examined the expression of relevant proteins. The FF-EtOAc suppressed the migration and quantity of EC cells in a dose-dependent relationship as compared to the blank group, according to the comparison of the aforementioned experimental data, and monitoring of MMP-9 expression revealed that FF-EtOAc suppressed MMP-9 expression (Fig. 4A-4D). Meanwhile, Transwell experiments demonstrate the same results (Fig. 4E).

Close

Fig. 4

Cell migration in EC cells is inhibited by FF-EtOAc. (A and B) In order to determine the effect of FF-EtOAc on the migration of Eca-109 cells, scratch wound healing experiments and Western blotting using β-actin as an internal control were employed to evaluate the expression levels of cell metastasis-related proteins. (C and D) In order to determine the effect of FF-EtOAc on the migration of KYSE-150 cells, scratch wound healing experiments and Western blotting using -actin as an internal control were employed to evaluate the expression levels of cell metastasis-related proteins. (E) In Transwell chambers treated with FF-EtOAc for 24 h, Eca-109 and KYSE150 cells were added, and the number of migrating cells was then counted. The control was β-actin. *P < 0.05, **P < 0.01 or ***P < 0.001 compare to control.

Export to PPT

3.4 Effects of FF-EtOAc on apoptosis of EC cells

The apoptotic effect of FF-EtOAc on EC cells cultivated in vitro was evaluated using Annexin V-FITC/PI, Hoechst 33258, and flow cytometry. After treated with FF-EtOAc for 12 h, EC cells have chromatin compression in their nuclei and the presence of apoptotic body, which are characteristic manifestations of apoptosis. And the number of cells with these characteristics increases with increasing dose (Fig. 5A). Results from flow cytometry and Annexin V-FITC/PI analysis demonstrated that FF-EtOAc caused EC cells to apoptosis in a dose-dependent way (Fig. 5B and 5C).

Close

Fig. 5

FF-EtOAc induced apoptosis in EC cells. (A) Eca-109 cells and KYSE-150 cells were incubated with 0, 10, 20, and 30 μg/mL for 12 h and stained with Hoechst 33258. (B) Eca-109 cells and KYSE-150 cells were exposed to 0 and 30 μg/mL for 12 h before being stained with Annexin V/PI and examined under a fluorescence microscope. (C) Eca-109 cells and KYSE-150 cells were exposed to 0 and 30 μg/mL for 12 h. After Annexin V-FITC and PI labeling, the fraction of apoptosis was determined by flow cytometry.

Export to PPT

3.5 Mitochondrial apoptosis and PI3K/Akt/Bad pathways are the main pathways of FF-EtOAc induction of apoptosis in vitro

F. ferulaeoides has been reported to inhibit cancer cell proliferation and promote apoptosis through the mitochondrial pathway and downregulate the expression of phosphorylated Akt (Kim et al., 2011, Zhang et al., 2015). In addition, downregulation of phosphorylated Akt expression phosphorylates downstream GSK-3 α/β and Bcl-2-associated death promoter (Bad) proteins, thereby preventing cancer cells from undergoing apoptosis (Zhang et al., 2020, Huang et al., 2021).

Western blot analysis was utilized to measure the expression levels of proteins associated with the mitochondrial apoptosis pathway and PI3K/Akt/Bad pathway. The results specifically showed that FF-EtOAc lowered the ratio of B-cell lymphoma-24(Bcl-2)/Bcl-2-associated X protein (Bax) and elevated the expression of cleaved Caspase-9, −3, and cleaved PARP in a dose-dependent way (Fig. 6A and 6B). The results also showed that FF-EtOAc concentration-dependently down-regulated the expression of p-Akt, p-GSK-3, and p-Bad, then elevated the expression of PTEN, but did not affect the protein levels of total Akt, GSK-3α/β, or Bad (Fig. 6C and 6D). All the results demonstrated that FF-EtOAc activated the mitochondrial and PI3K/Akt/Bad apoptotic pathways to cause apoptosis in vitro.

Close

Fig. 6

In vitro, mitochondrial apoptotic and PI3K/Akt/Bad pathways were used by FF-EtOAc to induce apoptosis (A and B). By Western blot assay, the relative expression of Bcl-2/Bax, cleaved caspase-9 and caspase-3, and PARP was detected after FF-EtOAc treated laryngeal carcinoma cells (0, 10, 20, and 30 μg/mL) for 12 h. (C and D) Using Western blot assay, the relative expression of PTEN, p-Akt/Akt, p-Gsk-3/Gsk-3 and p-Bad/Bad was detected after FF-EtOAc treated laryngeal carcinoma cells (0, 10, 20, and 30 μg/mL) for 12 h. The control was β-actin. *P < 0.05, **P < 0.01 or ***P < 0.001 compare to control.

Export to PPT

3.6 Effects of FF-EtOAc on growth inhibition in Eca-109 xenograft tumor nude mice and on apoptosis of Eca-109 cells in vivo

Female BALB/c nude mice were used to create Eca-109 subcutaneous tumor xenograft models, which were used to assess the therapeutic effectiveness of FF-EtOAc in vivo. When mice were treated with FF-EtOAc for 21 days, Eca-109 xenograft growth, volume, and weight were suppressed in dose-dependent way, but mice’s weight did not significantly change (Fig. 7A and 7B).

Close

Fig. 7

Apoptosis and the inhibitory effect in vivo of FF-EtOAc on transplanted tumors. (A and B) After treated with FF-EtOAc (0, 60 and 120 mg/kg) for 21 days, then We dissected tumors from the control and FF-EtOAc-treated mice. The tumor volume, tumor weight, and mouse weight were also counted. (C) Prepared tumor slices underwent TUNEL analysis. The green fluorescence stains in the Tunel experiment showed that the FF-EtOAc-treated mice's tumors underwent apoptosis. Eca-109 inoculated mouse tumor tissue samples were produced and IHC-tested. Manifestations of cleaved Caspase-3 and p-Akt were pictured and quantified by IHC. *P < 0.05, **P < 0.01 or ***P < 0.001 compare to control.

Export to PPT

Evaluation of the effect of FF-EtOAc on apoptosis production in vivo by TUNEL assay and IHC assay. TUNEL labeling of xenograft sections shows that the nuclei of apoptotic cells appear in fluorescent green and their number increases gradually in a dose-dependent manner (Fig. 7C). Meanwhile, the immunohistochemical assay results show that FF-EtOAc down-regulated the expression of p-Akt, but increased the expression of cleaved Caspase-3, (Fig. 7C). As per the results from HE staining, FF-EtOAc treatment caused chromatin agglutination and loosen the structure of tumor cells in a dose-dependent manner (Fig. 7C). Additionally, western blot analysis demonstrated that FF-EtOAc caused apoptosis in vivo activating the PI3K/Akt/Bad and mitochondrial apoptotic pathways, these results were shown by in vitro (Fig. 8A and 8B). Schematic summary of the mechanisms of FF-EtOAc induced EC cells apoptosis by mitochondrial and PI3K/Akt/Bad pathways in the present study (Fig. 9).

Close

Fig. 8

In vivo, FF-EtOAc caused apoptosis via the PI3K/Akt/Bad and mitochondrial apoptotic pathway. (A) By using Western blot technique, it was possible to identify the relative expression of Bcl-2/Bax, cleaved caspase-9 and caspase-3, and PARP in malignancies. (B) By Western blot assay, the relative expression of PTEN, p-Akt/Akt, p-Gsk-3/Gsk-3, and p-Bad/Bad was detected from tumors. The control was β-actin. *P < 0.05, **P < 0.01 or ***P < 0.001 compare to control.

Export to PPT

Close

Fig. 9

Schematic summary of the mechanisms of FF-EtOAc induced EC cell apoptosis in the present study.

Export to PPT