Role of Episamarcandin in Promoting the Apoptosis of Human Colon Cancer HCT116 Cells through the PI3K-Akt Signaling Pathway

This study identifies the active ingredients of Ferula sinkiangensis and investigates the role and mechanism of episamarcandin in colon cancer cells. The silica gel column chromatography was utilized to separate the chemical components of Ferula sinkiangensis. Sephadex LH-20 and semipreparative HPLC were adopted for further separation and purification. The compound episamarcandin showed good anticolon cancer activity among the 13 monomeric compounds obtained. Its effects on the apoptosis, cell cycle, and invasion and migration of colon cancer HCT 116 cells and PI3K-Akt signaling pathway were further investigated. The results showed that, similar to positive control cisplatin, episamarcandin inhibited the proliferation, promoted the apoptosis, arrested cells at G0/G1 phase, and suppressed migration and invasion of HCT 116 cells. A large number of apoptotic HCT 116 cells were observed under a transmission electron microscope. Fluorescence real-time quantitative PCR and western blot analysis showed that episamarcandin increased the expression of PTEN, p53, and Bax and decreased the expression of P-Akt, Akt, mTOR, Bcl-xl, and Bcl-2. Conclusively, episamarcandin may inhibit cell proliferation, migration, and invasion and promote the apoptosis of human colon cancer HCT 116 cells possibly through the PI3K-Akt signaling pathway.


Introduction
Ferula (about 150 species) belongs to Peucedaneae of Apiaceae, mainly distributed in Central Asia, such as Iran, Pakistan, Turkey, and the former Soviet Union. ere are 31 species in China [1].
e Chinese Pharmacopoeia (2015 edition) includes the resin of Ferula sinkiangensis or Ferula fukanensis [2]. Ferula sinkiangensis is mainly distributed in Yining County of Xinjiang and is endemic to China.
Uygur and Kazakh people in Xinjiang, China, have used the root or oleoresin Asafoetida to treat cancers, such as esophageal cancer, colon cancer, and uterine cancer [3]. In the past 20 years, plants of Ferula have attracted much attention due to their anti-inflammatory, anticancer, antiangiogenic, anti-P-glycoprotein, and antiviral activities. Studies have reported that plants of Ferula can inhibit proliferation and induce apoptosis and cell cycle arrest in human colon cancer HCT 116 cells, human glioma U87 cells, Raji lymphoma cells, cervical adenocarcinoma HeLa cells, and breast cancer MCF-7 cells as well as reducing the metastasis of breast cancer to lungs, livers and kidneys in animal models [4][5][6]. Our research group has also reported the antitumor and antioxidant activities of Ferula sinkiangensis [7][8][9][10]. We have demonstrated that the ethyl acetate fraction of Ferula sinkiangensis plays important role in inhibitory effect on colon cancer cells in vitro and in vivo. However, the specific antitumor active ingredients and mechanism of action remain unclear. e PI3K-Akt signaling pathway plays a central role in promoting cell proliferation, cell movement, invasion, and metastasis, inhibiting cell apoptosis and improving cell hypoxia tolerance [11,12]. e PI3K-Akt signaling pathway is activated when stimulated by extracellular signals such as cytokines, growth factors, and hormones. en, the downstream signaling molecule Akt is phosphorylated, which further activates or inhibits its downstream target proteins. PI3K -Akt pathway is usually overactivated in tumor tissues, leading to abnormal proliferation of tumor cells and a decrease in the number of apoptosis [13]. PI3K-Akt pathway can also promote the formation of vascular endothelial cells and affect the resistance of tumors to drugs [14,15].
Herein, we first identified the active ingredients of Ferula sinkiangensis by silica gel chromatography, Sephadex LH-20, and semipreparative high performance liquid chromatography (HPLC). Cisplatin, a widely used therapeutic drug for cancer, was used as a positive control in this study. MTT and SRB (sulforhodamine B) methods were used to verify the inhibitory effect of the 13 obtained monomer compounds on the proliferation of human colon cancer HCT 116 cells, which showed that the sesquiterpene coumarin compound episamarcandin had a significant inhibitory effect on HCT 116 cells. en, we evaluated the role and mechanism of episamarcandin in colon cancer cells. Cell proliferation, migration and migration, apoptosis, and cell cycle of HCT116 cells after treatment with episamarcandin were assessed. e apoptotic cell morphology was observed under a transmission electron microscope. Fluorescence real-time quantitative PCR and western blot were used to detect the expression levels of key genes of PI3K-Akt signaling pathway and apoptosis-related proteins. Our findings support that episamarcandin may serve as a potential alternative drug for the treatment of colon cancer.

Cell Line.
Human colon cancer HCT 116 cells were purchased from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). ey were cultured in RPMI l640 medium (HyClone).

SRB
Assay. Cells were treated as described in MTTassay. After treatment, 0.4% SRB was added and incubated for 30 min. After rinsing four times with 1% acetic acid, 150 μL Tris-base was added and incubated for 10 min on a shaker. e OD at 510 nm was determined using a microplate reader (Multiskan Spectrum, ermo Scientific, USA). Finally, calculation of cell inhibition rate (inhibition rate (%) � (OD (control group)-OD (episamarcandin group))/ (OD (control group)-OD (blank group)) × 100%) and IC 50 was performed.

Wound Healing Assay.
HCT 116 cells were plated into a 6-well plate at the concentration of 1 × 10 5 cells•mL −1 . An autolaved 10 μL sterile pipette tip was used to scratch lightly on the bottom of the culture plate three times per well. e scratch distance (width between scratch lines) at 0 h was recorded under an inverted microscope (IX-71, Olympus, Tokyo, Japan). Episamarcandin (50 μg•mL −1 ) and the positive control cisplatin (30 μg•mL −1 ) (Jiangsu Hansoh Pharmaceutical Group Co., Ltd., China) was added to each well. At 24 h and 48 h of incubation, the scratch distance (width between scratch lines) was measured, respectively.

Flow Cytometry Analysis.
HCT 116 cells were seeded in a 6-well plate at the concentration of 1 × 10 5 cells•mL −1 and cultured for 24 h. For cell apoptosis analysis, episamarcandin (50 μg•mL −1 , 25 μg•mL −1 and 12.5 μg•mL −1 ) and cisplatin (30 μg•mL −1 ) were added and incubated for 24 h. e cells were collected, resuspended, and incubated with Annexin V-FITC and PI (abs50001-100T, Absin, Shanghai, China). Cell apoptosis was analyzed on the flow cytometer (Guavaeasycyte 8HT, Millipore, USA). e total apoptosis rate was defined as the sum of the early apoptosis rate and late apoptosis rate. Evidence-Based Complementary and Alternative Medicine For cell cycle analysis, cells were cultured with episamarcandin (50 μg•mL −1 ) and cisplatin (30 μg•mL −1 ) for 24 h. e cells were collected and fixed with 75% ethanol at 4°C overnight. en, cells were incubated with RNase and PI for 30 min at 4°C in the dark. e flow cytometer (Guava easyCyte 8HT, Millipore, USA) was used to analyze cell cycle.

Morphology Observation with Transmission Electron
Microscope. HCT 116 cells were seeded in a 6-well plate at the concentration of 1 × 10 5 cells•mL −1 . After culture for 24 h, episamarcandin (50 μg•mL −1 ) and cisplatin (30 μg•mL −1 ) were added. After incubation for 24 h, the cells were collected and fixed with 2.5% glutaraldehyde. After rinsing, the sample was dehydrated with acetone, treated with 1% osmium acid at room temperature for 2 h, embedded in epoxy resin, cut into ultrathin sections, and stained with lead-uranium. Finally, the sample was observed under an electron microscope (Tecnai G2 Spirit 120 kV, ermo Scientific, USA). e primer sequences for EGFR (epidermal growth factor receptor), PTEN (phosphatase and tensin homolog deleted on chromosome ten), PI3Kp85, Akt1, mTOR (mammalian target of rapamycin), Bcl-xl, p53, Bcl-2, Bax, PI3Kp110δ, and β-actin are shown in Table 3. Fluorescence real-time quantitative PCR was performed on ABI7500 system (7500 Fast Real-Time PCR System, ABI, USA). Prepared cDNA sample was placed in a water bath at 50°C for 2 min, and in another water bath at 95°C for 2 min. After predenaturation, the cDNA sample was moved in a water bath at 95°C for 15 sec and in another water bath at 60°C for 60 sec. en, the sample was transferred to the PCR amplification machine for 30 cycles in total. e 2 −△△Ct method was used to calculate the level of each gene. β-Actin was used as internal reference.

Statistical
Methods. SPSS17.0 statistical software was used with all data expressed as mean ± standard deviation. One-way ANOVA was used for multiple comparisons followed by LSD method for pairwise comparison. Independent sample t-test was used to compare the differences Evidence-Based Complementary and Alternative Medicine 3 between two groups. P < 0.05 indicates that the difference is statistically significant.

e 13 CNMR Spectrum and 1 H-NMR Spectrum Parameters of Episamarcandin.
e chemical structure of episamarcandin was analyzed by 13 CNMR spectrum and 1 H-NMR spectrum. e structure of active compound episamarcandin was identified and shown in Figure 1 (Table 4).

Episamarcandin Inhibits the Proliferation of the Human Colon Cancer HCT116 Cells.
e MTT and SRB methods were used to determine the effect of episamarcandin on proliferation of HCT116 cells. e IC 50 of episamarcandin was determined as 26.06 μg•mL −1 and 29.71 μg•mL −1 , respectively. After 24 h of episamarcandin intervention, the cell shapes gradually changed from spindle shapes to oval ones (Figures 2(a) and 2(b)). Some of them were detached and became suspended cells. Statistically, the inhibition rate of HCT 116 cells was significantly increased with increased drug concentration, suggesting that the proliferation rate   (Figures 2(a) and 2(b)). is result implies that episamarcandin could inhibit proliferation of human colon cancer cells.

Episamarcandin Inhibits Migration of the Human Colon
Cancer HCT116 Cells. Cell migration was assessed with wound healing assay. After 24 h and 48 h of episamarcandin incubation, the width between scratch lines of the control group was shorter than that of episamarcandin and cisplatin groups (Figure 2(b)), suggesting that the wound healing degree of episamarcandin and cisplatin group was lower. e 24 h scratch distance of episamarcandin group and normal group was 407.3 ± 5.4 nm and 257.06 ± 7.6 nm, and the 48 h scratch distance was 380.3 ± 6.3 nm and 147.0 ± 6.5 nm, respectively. e migration rate of the episamarcandin group slowed down significantly compared with the control group (P < 0.05). e results suggest that episamarcandin has an inhibitory effect on the migration of HCT 116 cells.

Episamarcandin Promotes Apoptosis of the Human Colon
Cancer HCT116 Cells. After 24 h of episamarcandin intervention, total apoptotic rate of human colon cancer HCT116 cells was analyzed by flow cytometry. Compared with control group, the total apoptotic rate of the group treated with episamarcandin at the concentration of 12.5 μg•mL −1 , 25 μg•mL −1 , and 50 μg•mL −1 was significantly higher (Figure 3(a)) (P < 0.01).
us, episamarcandin could significantly promote the apoptosis of human colon cancer HCT116 cells.

Morphology and Structure Damage of HCT 116 Cells by
Transmission Electron Microscope. After treatment with episamarcandin (50 μg•mL −1 ) for 24 h, the cells were observed by transmission electron microscope (Figure 3(b)). e cells in control group were in round shape with many microvilli on the surface, evenly distributed organelles, large nuclei, complete nuclear membrane, and nucleolus. In   Evidence-Based Complementary and Alternative Medicine episamarcandin and cisplatin groups, the cells showed increased cytoplasmic density unevenly distributed around the cell membrane, incomplete cell membrane, invisible nucleoli, and many vacuoles and apoptotic bodies. e results showed that episamarcandin induced obvious apoptosis of human colon cancer HCT 116 cells.

Episamarcandin Arrests Cell Cycle of Human Colon
Cancer HCT 116 at G0/G1 Phase. After the intervention of episamarcandin, cell cycle of HCT 116 cells was analyzed with flow cytometry. Compared with control group, the number of cells in G0/G1 phase was significantly increased, and the number of cells in S and G2/M phases was significantly reduced in episamarcandin group (Figure 3(c)). is effect was similar to that of cisplatin group. e results indicate that episamarcandin could arrest the cell cycle of HCT 116 cells at G0/G1 phase.

Effect of Episamarcandin on HCT 116 Cell Migration and
Invasion. Transwell assays were applied to monitor the effect of episamarcandin on HCT 116 cell migration and invasion. e results showed that compared with normal control, episamarcandin (12.5 μg•mL −1 , 25 μg•mL −1 , and 50 μg•mL −1 ) and cisplatin had significantly lower numbers of migrated (Figure 4(a)) and invaded cells (Figure 4(b)).
is indicates that episamarcandin could inhibit the migration and invasion of HCT 116 cells.

Effect of Episamarcandin on Expression of Key Genes of PI3K-Akt Pathway.
e fluorescence real-time quantitative PCR was performed to detect the mRNA levels of key genes of PI3K-Akt pathway. Compared with the control group, the gene expression of EGFR and PI3Kp85 in HCT 116 cells after episamarcandin intervention did not change significantly (P > 0.05) ( Figure 5(a)). However, there was significant increase in expression of of p53, PTEN, PI3Kp110δ, and Bax genes (P < 0.05), while significant decrease in gene expression of Akt1, Bcl-xl, mTOR, and Bcl-2 (P < 0.05). In total, episamarcandin shows inhibitory effect on the PI3K/Akt signaling pathway.

Effect of Episamarcandin on Expression of Key Proteins of PI3K-Akt Pathway.
Based on the results of gene expression by fluorescence real-time quantitative PCR, the protein levels of PTEN, mTOR, Akt1, Bcl-xl, Bax, Bcl-2, and p53 with significant changes were further studied by western blot. Compared with the normal control group, the protein expressions of PTEN, p53, and Bax were significantly increased (P < 0.05), whereas those of Akt, p-Akt, Bcl-xl, mTOR, and Bcl-2 were significantly reduced (P < 0.05) in episamarcandin group (Figure 5(b)). e trend was consistent with that of fluorescent real-time quantitative PCR. is data further verified the inhibitory effect of episamarcandin on the PI3K/Akt signaling pathway.

Discussion
e cell-level drug screening model has the advantages of less material consumption, clear drug action mechanism, and large screening scale. In this study, the MTT method, SRB assay, scratch healing test, and Transwell assay were used to study the antitumor effect of the compound episamarcandin in vitro, which showed that the compound  32.0 (C-12′) 22 7.12 (1H, brs, H-8) 29.6 (C-13′) 23 17.1 (C-14′) 24 17.2 (C-15′) 6 Evidence-Based Complementary and Alternative Medicine Many studies have shown that inducing tumor cell apoptosis plays a major role in tumor treatment [16,17]. Transmission electron microscope has been adopted as the primary method to observe the morphology of apoptotic cells [18]. It can identify the fine structure smaller than 0.2 nm that cannot be observed under the optical microscope. In this study, under transmission electron microscope, the electron density of the cytoplasm of HCT 116 cells increased after the intervention with the compound episamarcandin and cisplatin with uneven thickness and incomplete cell membrane. e apoptotic bodies were observed. is observation showed that episamarcandin may induce the apoptosis of HCT 116 cells. Consistently, the results of flow cytometry in this study showed that the compound episamarcandin promoted the apoptosis of colon cancer HCT 116 cells.
Tumor cells are featured by immortal proliferation. erefore, to study cell proliferation and apoptosis, cell cycle has to be monitored. e results of this study showed that compared with control group, the number of HCT 116 cells in G0/G1 phase increased significantly, while that in the S phase and G2/M phase decreased significantly after intervention with episamarcandin and cisplatin.
us, episamarcandin arrested the cell cycle of HCT 116 cells at G0/G1 phase, thereby further inhibiting the proliferation of HCT 116 cells.
PI3K-Akt-mTOR signaling pathway is one of two downstream signaling pathways of EGFR, which can promote colon cancer cell proliferation, prolong cell survival, inhibit cell apoptosis, and participate in angiogenesis, leading to invasion and metastasis of colon cancer [19]. Our study showed that there was no statistically significant change in the expression of EGFR gene between the episamarcandin group and the control group. is suggests that episamarcandin may play a role in colon cancer HCT 116 cells in an EGFR independent way. e PI3K-Akt signaling pathway plays a vital regulatory role in cell proliferation and apoptosis [20]. It is usually overactivated in tumor tissues, leading to abnormal proliferation of tumor cells and reduced level of apoptosis [21]. PTEN can dephosphorylate PIP3 and inhibit PI3K activation, which negatively regulates the PI3K-Akt signaling pathway. It has been reported that the expression rate of PTEN in colon cancer tissues is significantly lower than that in normal mucosal tissues adjacent to the cancer, indicating that the decrease of PTEN is closely related to the proliferation and invasion of colon cancer cells [22]. Akt is also known as protein kinase B and is the main downstream signaling molecule of PI3K. Activated Akt further acts on its downstream target proteins through phosphorylation and then plays its role in regulating cell proliferation, differentiation, migration, and glucose metabolism [23,24]. TOR is one of downstream target genes of Akt. It has been reported that abnormal activation of mTOR can cause cell cycle activation, thereby promoting tumor formation and tumor cell invasion, metastasis and formation of blood vessels [25]. Bcl-2 family proteins are important components of the mitochondrial apoptosis pathway and play an important role in regulating the function of mitochondria and the release of cytochrome C.
e Bcl-2 family proteins mainly include three categories: antiapoptotic protein subfamily (Bcl-2, Bclxl, and Mcl-1); proapoptotic protein subfamily (Bax, Bak, and Bok); other proapoptotic proteins (Bid, Bim, and Puma). Bcl-xl has an independent effect on inhibiting cell apoptosis and can directly prevent apoptosis by interfering with the activity of caspase-3 [26]. Bax protein can combine with Bcl-2 to form dimers and coordinately regulate cell apoptosis. If the ratio of Bax/Bcl-2 is increased, Bax-Bax homodimers will increase, which in turn induces cell apoptosis [27]. e p53 gene is also a vital tumor suppressor gene. When the chromosomal DNA of a cell is damaged in the G1 phase, the transcription activity of p53 is enhanced to prevent further cell proliferation [28]. In addition, p53 can also enhance the arrest of tumor cell cycle by regulating other genes and proteins [29]. Our study showed that compared with the normal control group, the episamarcandin and cisplatin groups had significantly upregulated expression levels of PTEN, p53, and Bax, whereas they significantly downregulated expression levels Akt, Bcl-xl, mTOR, and Bcl-2.
It is reported that natural compounds such as calebin A and resveratrol can induce colon cancer cell apoptosis and prevent colorectal cancer metastasis by targeting SIRT1 and inhibiting NF-κB signaling [30][31][32]. Resveratrol can enhance the sensitivity of chemotherapeutics and improve the antitumor effect of FU on colorectal cancer cells by upregulating the cell-cell junctions, epithelial-mesenchymal transition, and apoptosis, inhibiting tumor necrosis factor-β signaling pathway and downregulating NF-κB [33,34]. Curcumin can enhance the 5-FU-induced reduction in the proliferation and invasion of colon cancer cells, which may help treat colorectal cancer and overcome drug resistance [35]. It is suggested that natural compounds may have chemotherapy sensitization potential and anticancer properties, and their combined use with chemotherapeutics can enhance their antitumor effects.
is study showed that episamarcandin had a good inhibitory effect on colon cancer, but whether it can be synergistically used in combination with cisplatin needs further study.
is study presents some limitations. For example, no in vitro or in situ experiments were performed. us, further studies are warranted for verification.

Conclusions
Our study demonstrates that episamarcandin could arrest cell cycle at the G0/G1 phase, inhibit proliferation, migration, and invasion, and promote the cell apoptosis of colon cancer HCT 116 cells. e underlying mechanism of episamarcandin may be upregulation of PTEN, p53, and Bax, reduction of Akt phosphorylation, and downregulation of mTOR, Bcl-xl, and Bcl-2, which may further lead to the inhibition of HCT 116 cell growth and proliferation and the promotion of apoptosis. Together, episamarcandin from Ferula sinkiangensis shows potentially promising inhibition effects against colon cancer HCT 116 cells and may be suitable as combination therapy or supportive therapy for colon cancer.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.