2.1. Antiproliferative Activity of Various Extracts
Figure 1 shows the anti-proliferative activity methanol and water extracts of CO leaves. The methanol extract, at concentrations above 1.25 µg/mL, showed anti-proliferative activities against HCT116 cells, whereas the water extract did not elicit marked activities because of the cell viability being greater than 90%.
Figure 1.
Antiproliferative activity of (a) methanol extracts and (b) water extracts of Chamaecyparis obtusa (CO) leaves (0–1.25 μg/mL) against HCT116 human colon cancer cells. Cells were treated with the extracts for 24 h. Data represent the mean of the percentage of control in triplicate tests. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate statistically significant difference compared to the control analyzed by the student’s t-test.
Figure 1.
Antiproliferative activity of (a) methanol extracts and (b) water extracts of Chamaecyparis obtusa (CO) leaves (0–1.25 μg/mL) against HCT116 human colon cancer cells. Cells were treated with the extracts for 24 h. Data represent the mean of the percentage of control in triplicate tests. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate statistically significant difference compared to the control analyzed by the student’s t-test.
The cell viabilities of the methanol and water extracts against Chang liver cells are shown in
Figure 2. Although the methanol extract significantly decreased the cell viability at 1.25 μg/mL, the cell viability remained above 90%. Therefore, MeOH extract of CO leaves demonstrated selective cytotoxicity against HCT116 human colon cancer cells, while it was ineffective in Chang liver cells. As described above, we observed antiproliferative activity with 1.25 µg/mL of methanol extract. Hence, this concentration of methanol extract was chosen for further studies.
It has previously been reported that epigallocatechin gallate and epigallocatechin showed antiproliferative and apoptotic activity against the HCT116 cell line at concentrations of 50–200 µM [
17]. Grape seed extract also showed anticancer activity against these cells at concentrations of 25–100 µg/mL [
18].
Artemisia herba has also been reported to show antiproliferative activity against the HCT116 cell line at concentrations of 25–200 µg/mL [
19]. Additionally, among the members of the Cupressaceae family, ethanol extract of
Juniperus phoenicea L. showed antiproliferative activity against HCT116 cells at concentrations of 5–50 µg/mL [
20]. Hinokitiol, isolated from
Thujopsis dolabrata, also has potential apoptotic activity against HCT116 colon cancer cells and SW620 cells at concentrations of 5–10 µM [
21]. The antiproliferative effects of widdrol, from
Juniperus chinensis, against the human colon adenocarcinoma cell line HT29, were also observed at concentrations of 16–64 µg/mL [
22]. Considering these previously published results, the methanol extract of CO leaves could represent a potent bioresource for the development of medicines with anti-colon cancer activity.
Figure 2.
Cell viability of (a) methanol extracts and (b) water extracts of Chamaecyparis obtusa (CO) leaves (0–1.25 μg/mL) against Chang liver cells. Cells were treated with the extracts for 24 h. Data represent the mean of the percentage of control in triplicate tests. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate statistically significant difference compared to the control analyzed by the student’s t-test.
Figure 2.
Cell viability of (a) methanol extracts and (b) water extracts of Chamaecyparis obtusa (CO) leaves (0–1.25 μg/mL) against Chang liver cells. Cells were treated with the extracts for 24 h. Data represent the mean of the percentage of control in triplicate tests. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate statistically significant difference compared to the control analyzed by the student’s t-test.
2.2. Comparative Global Metabolite Profiling of Methanol and Water Extracts Using GC-MS
PCA was performed to compare the metabolic profiles in methanol and water extracts of CO leaves. As shown in
Figure 3, there was a separation between methanol and water extracts of CO in the PCA-derived score plots mainly by principal component 1. The results imply that the metabolic profiles in methanol and water extracts of CO leaves varied according to the extraction solvents.
Comprehensive metabolic profiling of CO leaf extracts was performed using GC-MS to investigate the major compounds contributing to the anti-proliferative activity against HCT116 cells. As described in
Table 1, 16 metabolites in total were identified in the methanol and water extracts of CO leaves, and anthricin was identified in the methanol extract of CO leaf. The anthricin (deoxypodophyllotoxin) has been found in the dried root of
Anthriscus sylvestris, which is a wild plant from Europe, North America, Africa, and Asia [
23]. Anthricin has also been found in
Pulsatilla koreana [
24]. Anthricin has shown anticancer activity against leukemia, and prostate, breast, and uterine cervical tumors [
25,
26,
27,
28,
29,
30]. However, there have been no previous reports regarding the existence of anthricin in CO.
Figure 3.
Principal component analysis (PCA)-derived score plots of methanol and water extracts of Chamaecyparis obtusa leaves using five biological replicates.
Figure 3.
Principal component analysis (PCA)-derived score plots of methanol and water extracts of Chamaecyparis obtusa leaves using five biological replicates.
Table 1.
Relative intensity of various metabolites in the methanol (MeOH) and distilled water (DW) extracts of Chamaecyparis obtusa leaves. All data are presented as mean ± standard error of the mean of five replicates. * p < 0.05 (student’s t-test); ** p < 0.01 (Student’s t-test). RT, retention time; ND, not detected.
Table 1.
Relative intensity of various metabolites in the methanol (MeOH) and distilled water (DW) extracts of Chamaecyparis obtusa leaves. All data are presented as mean ± standard error of the mean of five replicates. * p < 0.05 (student’s t-test); ** p < 0.01 (Student’s t-test). RT, retention time; ND, not detected.
Compound | RT (min) | Relative Intensity |
---|
DW | MeOH |
---|
Alcohols | | | |
Glycerol * | 10.23 | 1.90 ± 0.23 | 1.19 ± 0.12 |
Mannitol * | 28.56 | 0.51 ± 0.07 | 0.28 ± 0.04 |
Myo-inositol * | 33.37 | 2.16 ± 0.23 | 1.12 ± 0.11 |
Amino acids | | | |
Aspartic acid ** | 16.58 | 0.06 ± 0.01 | ND |
Glutamic acid ** | 19.30 | 0.14 ± 0.02 | ND |
Serine ** | 12.47 | 0.05 ± 0.00 | ND |
Fatty acids | | | |
Palmitic acid ** | 32.25 | ND | 0.09 ± 0.01 |
Lignan | | | |
Anthricin ** | 57.14 | ND | 0.21 ± 0.02 |
Organic acids | | | |
Succinic acid * | 11.27 | 0.13 ± 0.02 | 0.08 ± 0.01 |
Malic acid ** | 15.80 | 4.61 ± 0.51 | 0.76 ± 0.08 |
Xylonic acid ** | 22.53 | 1.05 ± 0.10 | 0.25 ± 0.03 |
Phenolic acids | | | |
Shikimic acid * | 25.21 | 9.28 ± 1.03 | 5.73 ± 0.54 |
Sterol | | | |
β-Sitosterol ** | 57.92 | ND | 0.33 ± 0.03 |
Sesquiterpenes | | | |
Thujopsene ** | 14.44 | ND | 0.06 ± 0.01 |
β-Eudesmol ** | 22.64, 22.88 | ND | 4.03 ± 0.46 |
Sugar | | | |
Glucose * | 30.44 | 40.36 ± 11.01 | 20.15 ± 2.41 |
OPLS-DA was performed to obtain clearer separation of the two extracts and to reveal major compound contributing to the antiproliferative activity of CO leaf extracts. In
Figure 4, the S-plot clearly shows the separation of predictive variables between the methanol and water extracts. The S-plot visualizes the separation of various variables contributing to group separation. We observed the statistically significant metabolites from the methanol and water extracts using OPLS-DA (
Table 2). Various metabolites, such as alcohols, organic acids, sugar, and phenolic acid were identified in the two extracts, however, palmitic acid, thujopsene, anthricin, β-eudesmol and β-sitosterol were relatively higher in the methanol extract than in the water extract (
p < 0.01).
Figure 4.
S-plots obtained by orthogonal partial least-squares discriminant analysis (OPLS-DA) of methanol and water extracts of Chamaecyparis obtusa leaves.
Figure 4.
S-plots obtained by orthogonal partial least-squares discriminant analysis (OPLS-DA) of methanol and water extracts of Chamaecyparis obtusa leaves.
Table 2.
Metabolites identified in methanol (MeOH) and distilled water (DW) extracts of Chamaecyparis obtusa leaves separated by orthogonal partial least-squares discriminant analysis (OPLS-DA).
Table 2.
Metabolites identified in methanol (MeOH) and distilled water (DW) extracts of Chamaecyparis obtusa leaves separated by orthogonal partial least-squares discriminant analysis (OPLS-DA).
MeOH Extract (p < 0.01) | DW Extract (p < 0.05) |
---|
Palmitic acid | Glycerol |
Thujopsene | Mannitol |
Anthricin | Myo-inositol |
β-Sitosterol | Aspartic acid |
β-Eudesmol | Glutamic acid |
| Serine |
| Succinic acid |
| Malic acid |
| Xylonic acid |
| Shikimic acid |
We aimed to confirm if the anthricin contained in the methanol extracts of CO leaves has antiproliferative activity against HCT116 cells. We determined the amount of anthricin in the methanol extract of CO leaves as 36.1 μg/1000 mL (
Table 3). Based on the study, we investigated the antiproliferative activity of anthricin standard compound of various concentrations. As shown in
Figure 5, although the concentration of standard anthricin (1.6 μg/1000 mL) was 1/25 concentration of the anthricin content in methanol extract, the single anthricin standard showed similar antiproliferative activity of anthricin contained in methanol extract. Previous studies have also shown that crude extracts exhibit lower activity than sub-fractions, isolated compounds, or standards. The cranberry extracts showed lower activity than purified cyanidin glycosides in free radical scavenging activity, and the extracts of
Platycodon grandiflorum had a lower cytotoxicity on human cancer cells (HT-29, HRT-18 and HepG2) than the other sub-fractions and isolated compounds [
31,
32]. Thus, our result is in accordance with those previous reports, and we could speculate that various metabolites contained in the methanol extract might lower the antiproliferative activity of anthricin.
Table 3.
The regression equation and R2 value obtained from various concentration of anthricin, and the content of anthricin in the methanol extracts of C. obtusa leaves.
Table 3.
The regression equation and R2 value obtained from various concentration of anthricin, and the content of anthricin in the methanol extracts of C. obtusa leaves.
Compound | Regression Equation | R2 Value | Absolute Concentration a (μg/1000 mL) |
---|
Anthricin | y = 0.0029x − 0.6787 | 0.9945 | 36.1 |
Figure 5.
Effects of anthricin on cell proliferation in HCT116 human colon cancer cells. Cells were treated with anthricin (0–1.6 μg/1000 mL) for 24 h. Data represent the mean of the percentage of control in triplicate tests. ** p < 0.01 and *** p < 0.001 indicate statistically significant difference compared to the control analyzed by the student’s t-test.
Figure 5.
Effects of anthricin on cell proliferation in HCT116 human colon cancer cells. Cells were treated with anthricin (0–1.6 μg/1000 mL) for 24 h. Data represent the mean of the percentage of control in triplicate tests. ** p < 0.01 and *** p < 0.001 indicate statistically significant difference compared to the control analyzed by the student’s t-test.
2.3. Western Blot Analysis
Expressions of the apoptosis-related enzymes, caspase-3 and PARP (poly ADP ribose polymerase) were investigated using western blot analysis after exposure of cells to the methanol extract of CO leaves for 6 h to investigate the mechanisms underlying the cytotoxic effects of the extract. Caspase-3 is known as important mediator leading to the proteolytic cleavage of PARP, and causing apoptotic cell death [
33,
34].
As shown in
Figure 6a,b, the relative levels of cleaved caspase-3 and cleaved PARP were increased by methanol extract of CO leaves in dose-dependent manner. These results suggest that methanol extract of CO leaves might cause apoptotic cell death via a caspase-dependent pathway.
Figure 6.
Western blot of HCT116 cells treated with methanol extracts of Chamaecyparis obtusa leaves. (a) Representative western blot of caspase-3 and poly ADP ribose polymerase (PARP) protein expression; (b) Expression levels of caspase-3 and PARP protein. The level of each protein was normalized to beta-actin protein levels; (c) A representative western blot of phosphorylated-c-Jun N-terminal kinases (p-JNK) protein expression; (d) Expression levels of p-JNK protein. The results are from three independent experiments and presented as mean ± standard deviation. The letter labels on the graph indicate statistically significant differences between samples (p < 0.05) based on one-way ANOVA with Tukey’s post hoc test.
Figure 6.
Western blot of HCT116 cells treated with methanol extracts of Chamaecyparis obtusa leaves. (a) Representative western blot of caspase-3 and poly ADP ribose polymerase (PARP) protein expression; (b) Expression levels of caspase-3 and PARP protein. The level of each protein was normalized to beta-actin protein levels; (c) A representative western blot of phosphorylated-c-Jun N-terminal kinases (p-JNK) protein expression; (d) Expression levels of p-JNK protein. The results are from three independent experiments and presented as mean ± standard deviation. The letter labels on the graph indicate statistically significant differences between samples (p < 0.05) based on one-way ANOVA with Tukey’s post hoc test.
In addition, as shown in
Figure 7a,b, similar results were obtained in experiments performed with anthricin standard compound. Treatment of HCT116 cells with anthricin (0–1.6 μg/1000mL) caused significant increases in the levels of cleaved caspase-3 and cleaved PARP expression. These data indicate that the anthricin contained in the methanol extract of CO leaves induces apoptosis of HCT116 human colorectal cancer cell line.
Figure 7.
Western blot of HCT116 cells treated with anthricin (0–1.6 μg/1000mL). (a) Representative western blot of caspase-3 and poly ADP ribose polymerase (PARP) protein expression; (b) Expression levels of caspase-3 and PARP protein. The level of each protein was normalized to beta-actin protein levels. The letter labels on the graph indicate statistically significant differences between samples (p < 0.05) based on one-way ANOVA with Tukey’s post hoc test.
Figure 7.
Western blot of HCT116 cells treated with anthricin (0–1.6 μg/1000mL). (a) Representative western blot of caspase-3 and poly ADP ribose polymerase (PARP) protein expression; (b) Expression levels of caspase-3 and PARP protein. The level of each protein was normalized to beta-actin protein levels. The letter labels on the graph indicate statistically significant differences between samples (p < 0.05) based on one-way ANOVA with Tukey’s post hoc test.
Mitogen-activated protein kinase (MAPK) was known to be involved in cell proliferation, differentiation, apoptosis, and signaling cascades caused by external stimuli associated with tumor invasion and metastasis [
35,
36,
37]. MAPK includes three major enzymes: extracellular signal-regulated kinase (ERK), c-Jun
N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) [
38]. The expressions of JNK, ERK and p38 were analyzed by western blot analysis to investigate the contribution of those to apoptotic cell death of HCT116 by methanol extract of CO leaves. As shown in
Figure 6c,d, the level of the phosphorylated JNK (p-JNK) was increased in HCT116 cells by the treatment of methanol extract of CO leaves in dose-dependent manner. However, there were no increased phosphorylation of ERK and p38 (
Figure 8).
Figure 8.
Western blot of HCT116 cells treated with methanol extracts of C. obtusa leaves. Representative western blot of ERK and p38 protein expression.
Figure 8.
Western blot of HCT116 cells treated with methanol extracts of C. obtusa leaves. Representative western blot of ERK and p38 protein expression.
There have been several reports showing that the anti-proliferative activity of various agents such as resveratrol, butyrate, phenethyl isothiocyanate, and curcumin (diferuloylmethane) against colorectal cancer cell lines occurs through the JNK pathway, but not the ERK or p38 pathways [
39,
40,
41,
42]. As a stress-activated protein kinase, JNK phosphorylates c-Jun on Ser 63 and Ser 73 residues, and numerous studies indicate that JNK has a critical role in regulating cellular apoptosis factors [
43,
44]. JNK mediates the release of cytochrome c from mitochondria by modulating the activity of pro-apoptotic and anti-apoptotic mitochondrial proteins, such as the Bcl-2 family, and activates the caspase signaling cascade [
45,
46,
47,
48,
49,
50].
The level of p-JNK increased in response to treatment with the methanol extract of CO leaves, and it was presumed that this might cause mitochondrial dysfunction. Thereafter, the elevation of cleaved caspase-3 levels through cytochrome C activation induced the activation of PARP, which then lead to the apoptotic death of the HCT116 cells. Taken together, this study suggests that activation of p-JNK is a key event in the regulation of the apoptotic effect of the methanol extract of CO leaves on HCT116 cells. Our data provides evidence that the CO leaf extract might be an alternative anti-cancer agent that modulates the phosphorylation of JNK and induces apoptosis in human colon cancer cells.