Apoptosis is the process of programmed cell death, and induction of apoptosis is a therapeutic strategy in cancer treatment [1,2]. Apoptosis is occurred by various biological events, such as DNA damage and nutritional deficiency [2]. Therefore, regulation of major signaling pathways which mediate cell survival can be an important means of inducing apoptosis.

Akt (also called protein kinase B, PKB) plays an essential role in many cellular processes, including cell survival, growth, invasion, and metabolism [3]. Akt is activated by Ser473 phosphorylation and activation of Akt phosphorylates tuberous sclerosis complex 2 (TSC2) and glycogen synthase kinase-3β (GSK-3β) [4-6]. Direct phosphorylation of TSC2 by Akt stimulates cell survival and growth through activation of the mammalian target of rapamycin (mTOR) signaling pathway [5,7]. Also, the inhibitory form of GSK-3β (phosphorylated at Ser9) cannot regulate β-catenin protein stability, and β-catenin subsequently translocates to the nucleus and controls the expression of various genes, such as cyclin D1 and cyclooxygenase-2 (COX-2) [4,8,9]. COX-2 expression is controlled by both the Akt/mTOR and GSK-3β/β-catenin signaling pathways [10-13]. COX-2 inhibition leads to apoptosis through regulation of Bcell lymphoma 2 (Bcl-2) and pro-apoptotic proteins such as Bax and Bak, and induces G1 cell cycle arrest through decreasing cyclin D1 activity [10,14-16].

Regulation of Bcl-2 family proteins is very important in mitochondria-mediated apoptosis [17]. The anti-apoptotic protein, Bcl-2 suppresses pro-apoptotic proteins such as Bax and Bak, and, therefore, the Bax/Bak activation-mediated changes of mitochondrial membrane permeability (MMP) [17,18]. When cells receive apoptotic signals, Bax and Bak bind to each other and oligomerize to form pores in the mitochondrial outer membrane [19,20]. Following pore formation, cytochrome C and other molecules are released into the cytosol from the mitochondria and then apoptosis occurs by caspase-3 activation [21].

In this study, we determined the anti-tumor effects of Cnidium monnieri (L.) Cusson ethanol extracts in HT-29 colon cancer cells. C. monnieri has been used as a medicinal herb for the treatment of carbuncles, ringworm, nephritis, and gynecological diseases [22,23]. The bioactive components of Cnidium monnieri (L.) Cusson is coumarins, which includes osthole, xanthotoxin, isopimpinellin, bergapten, and imperatorin. Osthole, is one of the major coumarin compounds with various pharmacological functions, such as anti-cancer effects [24, 25].The anti-cancer effects of CME were determined in various cell lines [26-28]. However, research on the HT-29 cells is insufficient yet. To confirm the effects of CME-induced apoptosis and cell cycle arrest in HT-29 colon cancer cells, we investigated the regulation of the Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways, which are major signaling pathways involved in cancer progression.

2.1. Reagent

Cnidium monnieri (L.) Cusson were purchased from Dong Kyung PHARM (Seoul, South Korea). The 100 g of CME was soaked in 800 mL of 99.9% ethanol, and then stirring for 48 h at room temperature. The extract was filtered through filter paper (qualitative filter paper NO.1, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and concentrated with a rotary evaporator to remove the ethanol. The ethanol extracts of Cnidium monnieri (L.) Cusson (CME) was dissolved in DMSO (stock solution, 10~100 mg/mL) and refrigerated at −20°C. The final concentration of CME in the culture medium was controlled at 10~100 μg/mL. LY294002 (PI3K/Akt inhibitor) and BIO (GSK-3β inhibitor) were purchased from Calbiochem (San Diego, CA, USA) and Celecoxib (COX-2 inhibitor) and XAV939 (β-catenin inhibitor) was purchased from Sigma Aldrich (St., MO, USA).

2.2. Cell culture

HT-29 colon cancer cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were grown in RPMI medium (Hyclone, Laboratories Inc., Logan, UT, USA) containing 10% fetal bovine serum (Hyclone, Laboratories Inc., Logan, UT, USA) and 1% antibiotics (Hyclone, Laboratories Inc., Logan, UT, USA) at 37°C in a 5% CO₂ atmosphere. The cells were suspended by Trypsin-EDTA (Hyclone, Laboratories Inc., Logan, UT, USA) and separated at 1x10⁶cells/mL per 100-mm plate, every 48 h.

2.3. Determination of cell viability using MTT assay

The cells were seeded at 1 × 10⁴ cells/mL in 12-well plate and incubated for 24 h. Following incubation, the cells were treated with the CME (10~100 μg/mL) for 12, 24 or 48 h at 37°C in a 5% CO₂ atmosphere. The inhibitor was pre-treated for 30 min before treating with CME. The respective medium was removed, and cells were incubated with 20 μL of MTT solution (5 mg/mL) in phosphate- buffered saline (PBS) for 1 h. Converted purple formazan from MTT was solubilized in dimethyl sulfoxide (DMSO). The absorbance of the solution in each well was determined using a microplate reader (Model 680, Bio-Rad Laboratories, Inc., Tokyo, Japan) at 595 nm.

2.4. LDH release assay

The cells were seeded at 2.5 × 10⁵cells/mL per well in a 96-well plate, and incubated for 24 h. Following incubation, the cells were treated with the CME (20~100 μg/mL) and then incubated at 37°C in a 5% CO₂ atmosphere. The inhibitor was pre-treated for 30 min before treating with CME. After 24 h, the High control cells (Maximum LDH release) were treated with Cell Lysis Solution (Thermo Scientific, Rockford, IL, USA) for 45 min and then were centrifuged at 250 × g for 3 min (1730R, LaboGene, Seoul, South Korea). The absorbance of the solution in each well was determined using a microplate reader (Model 680, Bio-Rad Laboratories, Inc., Tokyo, Japan) at 490 and 655 nm.

2.5. Wound healing assay

The cells were seeded 2.5 × 10⁶cells/mL in a 6-well plate, and incubated for 24~48 h to form a cell monolayer. After the incubation a wound was made in the cell monolayer at center of well, and the cells were treated with CME at indicated does for 12 or 24 or 48 h at 37°C in a 5% CO₂ atmosphere. The wound was detected with a bright field microscope (Axioskop 50, Carl Zeiss, Thornwood, NY, USA).

2.6. Determination of apoptosis by Annexin V/PI double staining

The cells were seeded at 1 × 10⁶cells/mL in 60-mm plate and incubated for 24 h. Following incubation, the cells were treated with the CME for 24 h at 37°C in a 5% CO₂ atmosphere. The inhibitor was pre-treated for 30 min before treating with CME. Total cells were harvested by trypsinization, collected by centrifugation, washed with 3mL of PBS (twice), and resuspended in 1 mL of 1X Annexin-V binding buffer. Cells were stained with Annexin V and PI for 15 min. Fluorescence intensity was analyzed using a Flow cytometry-FACS Canto (Becton-Dickinson Biosciences, Drive Frankline Lage, NJ, USA).

2.7. Identification of apoptosis by Hoechst 33342

The cells were seeded at 1 × 10⁴cells/mL in 12-well plate and incubated for 24 h after put microscope cover glass (Marlenfeld GmbH & co. Germany) into well. Following incubation, the cells were treated with the CME (40, 60, 80 μg/mL) for 24 h at 37°C in a 5% CO₂ atmosphere. After 24 h, the cells were treated with 0.7 μM Hoechst 33342 and incubated for 30 min. Cells were fixed with 3.5% formaldehyde 0.5 mL for 20 min and then were gently washed three times with 150 μL of PBS for 5 min. Placed 10 μL of the mounting solution (50% glycerol) on a slide glass and covered with a cover glass. The chromatin was observed using fluorescence microscope (x200, Axioskop 50, Carl Zeiss, Thornwood, NY, USA).

2.8. Measurement of cell cycle arrest

The cells were seeded at 1x10⁶cells/mL in 60-mm plate and incubated for 24 h. Following incubation, the cells were treated with the CME for 24 h at 37°C in a 5% CO₂ atmosphere. The inhibitor was pre-treated for 30 min before treating with CME. Total cells were harvested by trypsinization, collected by centrifugation, washed with 3 mL of PBS (twice). The supernatant was removed and discarded. The pellet were resuspended in 1 mL of cold 70% ethanol and freezed at −20°C for at least 3 h. Ethanol-fixed cells were centrifuged at 3,000 rpm for 5 min and washed with 1 mL of PBS. The supernatant was removed, and ethanol-fixed cells were resuspended 0.5 mL of PBS. The cells were stained with 4 μl of PI (5 mg/mL) and 10 μl of RNase (10 mg/mL) for 20 min at room temperature. Fluorescence intensity was analyzed using a Flow cytometry-FACS Canto (Becton-Dickinson Biosciences, Drive Frankline Lage, NJ, USA).

2.9. Western blot analysis

The cells were seeded at 1 × 10⁵cells/mL in 6-well plate and incubated for 24 h. Then, the cells were treated with the concentration of CME for 24 h at 37°C in a 5% CO₂ atmosphere. The inhibitor was pre-treated for 30 min before treating with CME. The cells were then rinsed twice with ice-cold PBS and scraped with RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF) and subjected to Western blot analysis. Protein quantification was performed by Bradford assay. 30 μg of protein was loaded per lane. The Nitrocellulose membranes (0.45 μm; cat. no. 10600003; GE healthcare life science, Germany) were blocked with 2% bovine serum albumin (BSA, Bovogen, Melbourne, Australia) in 1X TBST (24.7 mM Tris-HCl, pH 8.0, 137 mM NaCl, 0.05% Tween-20) for 1 h 30 min and incubated overnight at 4°C with primary antibodies targeting mouse monoclonal-p-Akt (Ser473) (1:2,000; cat. no. 4051), rabbit monoclonal-Akt (1:1,000; cat. no. 4685), rabbit monoclonal-GSK-3β (1:1,000; cat. no. 9315), rabbit monoclonal-p-TSC2 (Thr 1462) (1:1,000; cat. no. 3617), rabbit monoclonal-TSC2 (1:1,200; cat. no. 3635), rabbit polyclonal-p-mTOR (Ser2448) (1:1,000; cat. no. 2917), rabbit polyclonal-mTOR (1:1,000; cat. no. 2972), rabbit polyclonal-β-catenin (1:500; cat. no. 9562), rabbit polyclonal-COX-2 (1:1,000; cat.no. 4842), rabbit polyclonal-Bcl-2 (1:2,000; cat. no. 2876), rabbit monoclonal-Bax (1:1,000; cat. no. 5023), rabbit monoclonal-Bak (1:1,000; cat. no. 6947), rabbit monoclonal-caspase-3 (1:1,000; cat. no. 9665), rabbit monoclonal-PARP (1:1,000; cat. no. 9532), and rabbit polyclonal-β-actin (1:2,000; cat. no. 4967); all purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse monoclonal-p-β-catenin (Ser 33/37) (1:1,000; cat. no. ab11350) and Rabbit polyclonal-COX-IV (1:1,000; cat. no. ab153709) were purchased from Abcam Inc. (Cambridge, MA, USA). Rabbit polyclonalp-GSK-3β (Ser9) (1:1,000; cat. no. sc-11757-R) and Mouse monoclonal-Cytochrome c (1:2,000; cat. no. sc-13156) were purchased from Santa Cruz Inc. (Santa Cruz, CA, USA). After primary antibody incubation, the membranes were washed 4 times for 5 min each with 1X TBST at room temperature. Following the addition of the secondary antibody; goat polyclonal-anti-mouse antibody conjugated with HRP (1:10,000; cat. no. PA1-30126; Thermo Scientific Rockford, IL, USA) and goat anti-rabbit antibody conjugated with HRP (1:10,000; cat. no. 166-2408; Bio-Rad Laboratories, Inc., Tokyo, Japan), the membrane were reacted for 1 h 30 min at room temperature with gentle agitation. After secondary antibody incubation, the membranes were washed 4 times for 10 min each with 1X TBST at room temperature. Proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (cat. no. PI34080; Thermo Scientific Rockford, IL, USA) and visualized on CP-BU new X-ray film (Agfa HealthCare, Inc., Mortsel, Belgium).

2.10. Caspase-3 activity assay

The cells were placed in 6-well plate at 1 × 10⁵cells/mL. Following incubation for 24 h, the cells were treated with CME and specific inhibitor. The Cells were harvested by Trypsin-EDTA (Hyclone, Laboratories Inc., Logan, UT, USA), and then incubated with 50 μl of chilled Cell Lysis Buffer on ice for 10 min. The lysates of 130 μg Proteins were added to Reaction Buffer with 10 mM DTT. After added 200 μM DEVDp-NA, incubate at 37°C for 1 h 30 min. The absorbance of the solution in each well was determined using a microplate reader (Model 680, Bio-Rad Laboratories, Inc., Tokyo, Japan) at 415 nm.

2.11. Fraction of mitochondria and cytosol proteins

We used Mitochondria/Cytosol Fraction Kit (ab65320, Abcam PLC., Cambridge, UK). Cells were seeded at 1 × 10⁶cells/mL in 100-mm plate and incubated for 24 h. After incubation, cells were treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Total cells were harvested by trypsinization, collected by centrifugation, washed with PBS, and homogenized in icecold cytosol extraction buffer mix containing DTT and protease inhibitor using a sonicator (KSS-650D, Korea Process Technology Co., Ltd., Seoul, South Korea). The homogenates were centrifuged at 3,000 rpm for 10 min at 4°C and supernatants were collection. Supernatant were centrifuged at 13,000 rpm for 30 min at 4°C and collected supernatant for cytosol proteins and pellets were resuspended with ice cold mitochondria extraction buffer containing DTT and protease inhibitor for mitochondria proteins.

2.12. Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted using RiboEx (GeneAll, GeneAll biotechnology, Seoul, South Korea) according to the manufacturer's instructions, and cDNA was generated using Reverse Aids cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. RTPCR was performed with the following temperature profile: a pre-denaturation step of 10 min at 95°C, followed by 35 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec and a final exposure at 72°C for 10 min. The RT-PCR was carried out using the specific primers: COX-2-F – 5’-TGAGCATCTACGG TTTGCTG-3’; COX-2-R – 5’-TGCTTGTCTGGAACAACTGC -3’; β-actin-F- 5’- GGACTTCGAGCAAGAGATGG-3’; β-actin-R- 5’- AGCACTGTGTTGGCGTACAG-3’. All primer sequences were designed using the Primer3 online program and synthesized by Bioneer Corp (Daejeon, South Korea). The PCR products were analyzed on 2% agarose gel and visualized using EcoDye™ DNA Staining Solution (BIOFACT Co., Ltd., South Korea).

2.13. Tumor formation (Xenograft model)

Five-week-old male Balb/c nu/nu mice were obtained from SLC (Tokyo, Japan) and housed in sterile filer-topped cages. For tumor induction, HT-29 human colon cancer cells (2.5 × 105cells/0.1 mL) were subcutaneously injected into the left flank of the mice (each group had 3 animals). One week after the injection of cells, they were co-treated with CME 60 and 80 mg/kg/day for 28 days. The concentration range of CME was determined through preliminary experiment. We each diluted 100 μl of 150 and 200 mg/mL CME in DMSO with 900 μl of PBS. Then, we subcutaneous injected 100 μl of 15 and 20 mg/mL of CME per day (60 and 80 mg/kg/day) in mice. Tumor size was measured using a caliper at 2 day intervals, and the volume was calculated by the modified formula V = 1/2 (length x width2). After the 4-week treatment, tumor was removed and frozen in liquid nitrogen for western blot analysis or fixed with formalin for immunohistochemistry and H&E staining.

2.14. Immunohistochemistry

Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Consecutive thin cryosections (5 μm) of OCT compound (Sakura Finetek, Torrance, CA, USA) embedded tumor tissues were fixed in acetone at 4°C for 10 min. After washing in PBS, sections were treated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity, and the sections were blocked with normal rabbit serum. Then, the sections were blocked and washed in PBS and incubated with specific antibody overnight at 4°C. Negative controls were incubated with the primary normal serum IgG for the species from which the primary antibody was obtained.

2.15. TUNEL assay

Levels of apoptosis in distal colon tissue were determined using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Tissue sections were processed according to manufacturer's instructions for the ApopTag peroxidase in situ apoptosis detection kit (Vector Laboratories, Burlingame, CA, USA).

2.16. Statistics

All data were expressed as arithmetic mean ± standard deviation. Statistical analysis was performed with unpaired a one-way ANOVA and independent sample t-test (IBM SPSS Statistics 20.0, SPSS Inc., Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

3.1. CME treatment inhibits cell proliferation and induces morphology changes in HT-29 cells.

Colon cancer consists of malignant tumors and has a high worldwide incidence; it is the second most common cancer in females, and the third most common in males [29]. The causes of cancer include excessive drinking, smoking, aging, and adaptation to Western eating habits [30]. Recently, the anticancer effects of various plant extracts have been focused on and reported [31-34]. Many previous studies reported that natural plant extracts suppress cell proliferation in colon cancer cells [35-38]. Moreover, the treatments of CME did not show cytotoxicity in normal cells such as Fibroblast and HaCaT cells [26,27]. We examined the anti-proliferative and cytotoxic effects of CME using the MTT assay and LDH release assay. Cells were treated with CME (10~100 μg/mL) for 12, 24, or 48 h. Our results showed that CME suppresses cellular proliferation and induces cytotoxicity in a dose-dependent manner Fig. 1(a) and (b). Also, cells appeared to exhibit anti-migration activity according to inhibition of cell proliferation by CME treatment Fig. 1(c). Moreover, we confirmed the morphological changes that occur in HT-29 colon cancer cells. Cell shrinkage and a decrease in cell proliferation were observed when cell were treated with CME (20~100 μg/mL) Fig. 1(d). These results indicated that CME treatment induces anti-proliferative, cytotoxic, and cell morphological changes in HT-29 colon cancer cells also like other studies [26-27, 35-38].

Figure 1. Cytotoxic and anti-proliferative effects of CME in HT-29 colon cancer cells. (a) Cells were treated with variable concentrations of CME (10~100 μg/mL) for 12 h, 24 h and 48 h. Cell viability was measured by MTT assay. The statistical analysis of the data was carried out by use of independent sample t-test. *,#p < 0.05, **,##p < 0.01 and ***,###,+++p < 0.001 vs. con (each experiment, n = 3). (b) LDH release was measured by LDH release assay. The statistical analysis of the data was carried out by use of a one-way ANOVA with Duncan multiple range test (p < 0.05). (c) Anti-migration effects of CME in HT-29 colon cancer cells. Cell were treated with variable concentrations of CME (20~100 μg/mL) for 24 h. Scratch wound healing assay was performed to examine the anti-migration effects of CME. (d) The cell morphology change after treatment of CME was observed by invert microscopes (x200 magnification, Axiovert 100, Zeiss, Germany). N.S., not significant; CME, Cnidium monnieri (L.) Cusson extract; con, control.

3.2. CME treatment induces apoptosis and cell cycle arrest at the G1 phase in HT-29 cells.

To determine whether CME-induced inhibition of cell viability and morphological changes occurred through apoptosis, we investigated Annexin-V/PI double staining and Hoechst 33342 staining after treatment with various concentration of CME (40~80 μg/mL) for 24 h. As shown in Fig. 2(a), (b) and (c), the percentage of Annexin-V-positive cells increased by 2.47 (control), 7.96 (40 μg/mL), 14.31 (60 μg/mL), and 15.71% (80 μg/mL) and apoptotic DNA fragmentation was increased in a dose-dependent manner. Furthermore, we confirmed the cell cycle arrest effect of CME and found that the concentration of G1-phase cells increased following treatment with CME Fig. 2(d) and (e). According to previous studies, it has been reported that CME induce anti-cancer effects through cell cycle arrest in various cancer cell lines [27-28]. Also, these results indicate that CME-induced cell death characteristics, such as a decrease in cell proliferation and morphological changes, occurred through apoptosis and cell cycle arrest in HT-29 colon cancer cells.

Figure 2. Apoptotic and cell cycle arrest effects of CME in HT-29 colon cancer cells. Cell were treated with variable concentrations of CME (40~80 μg/mL) for 24 h. (a) Apoptotic effects of CME were evaluated by Annexin V-PI double staining. (b) The graph of apoptosis rate. (c) Apoptotic bodies were measured by Hoechst 33342 staining. The chromatin was observed using fluorescence microscope (Axioskop 50, Carl Zeiss, Thornwood, NY, USA). Origin magnification x200. (d) CME occur cell cycle arrest at G1 phase. Cell cycle arrest effects were measured by flow cytometric. (e) The graph of cell cycle phase. CME, Cnidium monnieri (L.) Cusson extract.

3.3. CME treatment regulates expression of apoptosis-related proteins in HT-29 cells.

Akt phosphorylates TSC2 and GSK-3β and regulates activation of these protein-mediated signaling pathways [5,39]. Regulation of mTORC1 by phosphorylation of TSC2 induces cell survival and growth [5]. Also, inhibition of the Akt/mTOR signaling pathway induces decrease in COX-2 expression [14,40]. Furthermore, the inactive form of GSK-3β cannot degrade β-catenin, and its presence leads to β-catenin translocation to the nucleus. Nuclear β-catenin regulates COX-2 expression, because COX-2 is transcription targets of β-catenin/Tcf signaling [41-43]. So, COX-2 expression is regulated by Akt/mTOR and GSK-3β/β-catenin signaling pathway [10-13]. According to previous studies, COX-2 is overexpressed in colon cancer about 80-90% higher than normal colon mucosa [42-45]. Inhibition of COX-2 expression by natural compounds such as quercetin decreases Bcl-2 expression and induces Bax and caspase-3-dependent apoptotic signaling pathway [15,46]. Moreover, according to previous study, TSN treatment attenuated levels of p-AKT, p-GSK-3β, β-catenin as well as survivin, c-Myc and COX-2 which are β-catenin-activated genes in SW480 colon cancer cells [47]. We observed changes in the expression levels of apoptosis-related proteins after CME treatment by Western blot analysis in HT-29 colon cancer cells. Our results showed that the protein levels of pmTOR, p-TSC2, β-catenin, COX-2, p-Akt, and p-GSK-3β were decreased and the protein levels of p-β-catenin, Bax, and Bak were increased by CME treatment Fig. 3(a). Also, we confirmed not only a decrease in procaspse-3 expression but also an increase in the activity of caspase-3 with a caspase-3 activity assay Fig. 3(b). The translocation of apoptotic proteins plays a significant role in the mitochondrial-mediated apoptosis pathway. Therefore, we examined the expression levels of apoptotic proteins after fractionation of the mitochondria and cytosol. As shown in Fig. 3(c), expression of the mitochondrial anti-apoptotic protein Bcl-2 decreased and that of mitochondrial apoptotic proteins such as Bax and Bak were increased following CME treatment. Also, cytochrome C is released into the cytosol from the mitochondria.

Figure 3. CME regulate apoptosis-mediated proteins expression in HT-29 colon cancer cells. (a) CME effects on p-mTOR, mTOR, p-TSC2, TSC2, PARP, p-β-catenin, β-catenin, COX-2, p-Akt, Akt, p-GSK-3β, GSK-3β, procaspase-3, Bcl-2, Bak and Bax. Cells were treated CME (40~80 μg/mL) for 24 h. Protein levels were determined by Western blot analysis. The β-actin probe served as proteinloading control. (b) CME induces caspase-3 activation in HT-29 colon cancer cells. Cells were treated with CME (40~80 μg/mL) for 24 h. The statistical analysis of the data was carried out by use of independent sample t-test. **p < 0.01 vs. con (each experiment, n = 3). (c) Western blot analysis of Bcl-2, Bak, Cytochrome c, COX-IV, and β-actin in cytosolic and mitochondrial fractions of HT-29 cells treated with CME (40~80 μg/mL) for 24 h. The β-actin probe served as protein-loading control in cytosol. The COX-IV probe served as proteinloading control in mitochondria. N.S., not significant; CME, Cnidium monnieri (L.) Cusson extract; p, phosphorylated; mTOR, mammalian target of rapamycin; TSC2, tuberous sclerosis complex 2; PARP, Poly (ADP-ribose) polymerase; COX-2, cyclooxygenase-2; Akt, protein kinase B; GSK-3β, glycogen synthase kinase-3β; Bcl-2, B-cell lymphoma 2; Bak, Bcl-2-homologous antagonist killer; Bax; Bcl-2-associated X protein; con, control.

3.4. CME treatment induces apoptosis and cell cycle arrest through reducing COX-2 expression by regulating the Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways.

To identify whether the effects of CME-induced apoptosis and cell cycle arrest occurred through control of COX-2 expression by regulating not only Akt/GSK-3β/mTOR but also Akt/GSK-3β/β-catenin signalling pathways, we treated HT-29 colon cancer cells with LY294002 (PI3K/Akt inhibitor), BIO (GSK-3β inhibitor), XAV939 (β-catenin inhibitor), and Celecoxib (COX-2 inhibitor). The MTT assay revealed decreased cell viability in the LY294002-treated group compared to the control group, whereas, viability of the CME/BIO co-treated group was not decreased compared to the CME-treated group Fig. 4(a). Also, cell viability in the Celecoxib- or XAV939-treated groups decreased. Furthermore, LDH release in the LY294002-, celecoxib-, or XAV939-treated groups increased Fig. 4(b). LDH release did not significantly differ between the CME/BIO co-treated group and the control group. Annexin-V/PI double staining and cell cycle analysis were performed, to identify the influence on apoptosis and cell cycle of LY294002, BIO, XAV939, or celecoxib. The results showed that the percentage of Annexin-V positive cells increased by 11.83 (LY294002), 9.369 (celecoxib), and 10.37% (XAV939) compared to the control group (5.93%), while the number of Annein-Vpositive cells in the CME/BIO co-treated group did not significantly increase (6.02%) Fig. 4(c). Furthermore, the ratio of G1 phase cells increased by 70.44 (LY294002), 65.64(celecoxib), and 56.92% (XAV939) compared to the control group (48.9%), while the CME/BIO co-treated group did not show such an increase (48.83%) Fig. 4(d).

Figure 4. CME induces anti-proliferation effects, apoptosis and cell cycle arrest at G1 phase via regulating Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways. Cells were treated with 20 μM LY294002 or 1 μM BIO or 20 μM Celecoxib or 1 μM XAV939 or 60 μg/mL CME for 24 h. (a) Cell viability was measured by MTT assay. The statistical analysis of the data was carried out by use of independent sample t-test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. con. ##p < 0.01 and ###p < 0.001 vs. CME-treated group (each experiment, n = 3). (b) LDH release was measured by LDH release assay. The statistical analysis of the data was carried out by use of independent sample t-test. **p < 0.01 and ***p < 0.001 vs. con. #p < 0.05 and ###p < 0.001 vs. CME-treated group (each experiment, n=3). (c) Apoptotic effects of CME were evaluated by Annexin V-PI staining. (d) Cell cycle arrest effects were measured by flow cytometric. N.S., not significant; CME, Cnidium monnieri (L.) Cusson extract; con, control.

To confirm the regulation of the Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways, we investigated the expression levels of apoptosis-associated proteins following treatment of HT-29 colon cancer cells with LY294002, celecoxib, or XAV939. When we treated cells with LY294002, the expression of p-Akt, p-GSK-3β, p-TSC2, p-mTOR, β-catenin, and COX-2 decreased and the protein levels of p-β-catenin, Bax, and Bak increased, which is similar to the effects seen in the CME-treated group Fig. 5(a). p-Akt protein expression decreased while the other proteins were not affected in the CME/BIO co-treated group. Additionally, p-β-catenin, β- catenin, COX-2, and apoptosis-mediated proteins (Bcl-2, Bax, Bak, and pro-caspase-3) were regulated by XAV 939 treatment and p-β-catenin and β-catenin expression were not regulated by treatment with celecoxib treatment Fig. 5(b). The rate of caspase-3 activity was not significantly different between the CME/BIO co-treated and control groups Fig. 5(c). Also, we confirmed that COX-2 mRNA expression was regulated by β- catenin Fig. 5(d). These results support the notion that CMEinduced apoptosis and cell cycle arrest occurred via the Akt/ GSK-3β signaling pathway and COX-2 expression was regulated by both the Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways.

Figure 5. CME regulates apoptosis-mediated proteins through reducing COX-2 expression by regulating the Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways in HT-29 colon cancer cells. (a) Proteins expression of p-mTOR, mTOR, p-TSC2, TSC2, p-β-catenin, β-catenin, COX-2, p-Akt, Akt, p-GSK-3β, GSK-3β, procaspase-3, Bcl-2, Bak, and Bax. (b) Cells were treated with 20 μM Celecoxib or 1 μM XAV939 or 60 μg/mL CME for 24 h. Protein levels were determined by Western blot analysis. The β-actin probe served as protein-loading control. (c) Caspase-3 activity assay. The statistical analysis of the data was carried out by use of independent sample t-test. **p < 0.01 and ***p < 0.001 vs. con. ###p < 0.001 vs. CME-treated group (each experiment, n = 3). (d) CME, Celecoxib and XAV939 effects on COX-2 mRNA expression. Representative reverse-transcriptase-PCR pictures are shown with β-actin as the housekeeping gene. The statistical analysis of the data was carried out by use of independent sample t-test. ***p < 0.001 vs. con (each experiment, n = 3). N.S., not significant; CME, Cnidium monnieri (L.) Cusson extract; p, phosphorylated; mTOR, mammalian target of rapamycin; TSC2, tuberous sclerosis complex 2; PARP, Poly (ADP-ribose) polymerase; COX-2, cyclooxygenase-2; Akt, protein kinase B; GSK-3β, glycogen synthase kinase-3β; Bcl-2, B-cell lymphoma 2; Bak, Bcl-2-homologous antagonist killer; Bax; Bcl-2-associated X protein; con, control.

3.5. CME treatment induces apoptosis through the Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways in an HT-29 xenograft model.

To assess the influence of CME treatment in an HT-29 xenograft model, we performed histological experiments following treatment with two different concentrations of CME (60 and 80 mg/kg/day). We primarily identified that treatment with CME (60 and 80mg/kg/day) attenuated tumor proliferation compared to that seen in the control group, without causing a change in body weight Fig. 6(a). In addition, Akt/GSK-3β signaling proteins were regulated and apoptotic proteins were translocated by CME treatment in vivo Fig. 6(b), (c) and (d). Previous studies showed that tumor tissues are degraded and the concentration of TUNEL-positive cells increased following treatment with natural extracts or compounds [48-50]. HT-29 xenograft tumor tissue was degraded and the number of TUNEL-positive cells was increased in the CME-treated group Fig. 7(a). In preceding research, protein expression of p-GSK-3β and β-catenin expression levels were reduced owing to treatment with an anti-cancer agent in a xenograft model [51,52]. Using immunohistochemical analysis, we determined that the control group showed higher expression of p-GSK-3β and β-catenin and the CME (60 and 80 mg/kg/day)-treated groups were showed reduced expression of cytosolic p-GSK-3β and nuclear β-catenin compared to the control group Fig. 7(b).

Figure 6. CME regulates tumor growth and expression of apoptosis-mediated protein in a HT-29 xenograft model. (a) HT-29 colon cancer cells (1 × 10⁶ cells/0.1 mL) were injected subcutaneously into the left flanks of Balb/C nu/nu mice (n = 3 per group). After 1 week, mice received CME subcutaneous (SC) (60 and 80 mg/kg/day) for 28 days. Tumor volume was measured once every 2 days and calculated as described in the Materials and Methods section. Body weight was measured once each week. Photographs showed tumor xenograft morphologies in the various groups. (b) Protein expression levels of apoptosis-mediated proteins in vivo. (c) Caspase-3 activity assay. The statistical analysis of the data was carried out by use of independent sample t-test. *p < 0.05 and ***p < 0.001 vs. con (each experiment, n = 3). (d) Western blot analysis of Bcl-2, Bak, Cytochrome c, COX-IV, and β-actin in cytosolic and mitochondrial fractions of HT-29 xenograft model. The β-actin probe served as protein-loading control in cytosol. The COX-IV probe served as protein-loading control in mitochondria. CME, Cnidium monnieri (L.) Cusson extract; Bcl-2, B-cell lymphoma 2; Bak, Bcl-2-homologous antagonist killer; Bax; Bcl-2-associated X protein; con, control.

Figure 7. Effects of CME on p-GSK-3β and β-catenin expressions in vivo. (a) H&E staining and TUNEL assay of apoptosis cells in tumor tissue. (b) The levels of p-GSK-3β and β-catenin were also measured by immunohistochemical analysis. The slides were observed under a microscope (magnification, x100 and x400) and images were captured. Black circle indicate cytosolic p-GSK-3β and yellow arrows indicate nuclear accumulation of β-catenin. CME, Cnidium monnieri (L.) Cusson extract; H&E, Hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling; GSK-3β, glycogen synthase kinase-3β.

In our study, we identified the apoptosis and cell cycle arrest effects of an extract from the fruit of C. monnieri in HT-29 colon cancer cells. We indicated that CME treatment induces anti-proliferative, cytotoxic, and cell morphological changes in HT-29 colon cancer cells. Also, our results demonstrated that CME-induced apoptosis and G1 cell cycle arrest effects occurred by regulation of COX-2 expression through control of Akt/GSK-3β/mTOR as well as Akt/GSK-3β/β-catenin signaling pathways. In conclusion, a CME-induced anti-cancer effect such as apoptosis occurred via a mitochondria-mediated pathway that is down-regulated by COX-2 expression. Also, this CMEinduced decrease in COX-2 expression is controlled by both Akt/GSK-3β/mTOR and Akt/GSK-3β/β-catenin signaling pathways.

This work was supported by 2022 Hannam University Research Fund. All animal experiments were approved by the ethics committee for animal experimentation, hannam university (HNU 2016-8).