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The Antiproliferative Effect of Chakasaponins I and II, Floratheasaponin A, and Epigallocatechin 3-O-Gallate Isolated from Camellia sinensis on Human Digestive Tract Carcinoma Cell Lines

Acylated oleanane-type triterpene saponins, namely chakasaponins I (1) and II (2), floratheasaponin A (3), and their analogs, together with catechins—including (–)-epigallocatechin 3-O-gallate (4), flavonoids, and caffeine—have been isolated as characteristic functional constituents from the extracts of “tea flower”, the flower buds of Camellia sinensis (Theaceae), which have common components with that of the leaf part. These isolates exhibited antiproliferative activities against human digestive tract carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 cells. The antiproliferative activities of the saponins (1–3, IC50 = 4.4–14.1, 6.2–18.2, 4.5–17.3, and 19.3–40.6 µM, respectively) were more potent than those of catechins, flavonoids, and caffeine. To characterize the mechanisms of action of principal saponin constituents 1–3, a flow cytometric analysis using annexin-V/7-aminoactinomycin D (7-AAD) double staining in HSC-2 cells was performed. The percentage of apoptotic cells increased in a concentration-dependent manner. DNA fragmentation and caspase-3/7 activation were also detected after 48 h. These results suggested that antiproliferative activities of 1–3 induce apoptotic cell death via activation of caspase-3/7.


Antiproliferative Activities of Constituents Isolated from "Tea Flower" against Human Gastric Carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 Cells
Digestive tract carcinoma refers to a group malignancy located in the oral cavity, pharynx, larynx, esophagus, stomach, and large intestines and is the most common type of cancer worldwide [13]. In our previous studies of bioactive components from natural medicines isolated from the flowers of Bellis perennis, we observed antiproliferative activities of acylated oleanane-type triterpene saponins, perennisaponins A-T, against HSC-2, HSC-4, and MKN-45 cells [14]. The structures of perennisaponins resemble that of saponin constituents isolated from C. sinensis, and thus, similar activities were expected. Two analytical protocols have been developed with respect to nine acylated oleanane-type triterpene saponins, including chakasaponins I (1), II (2), and III, floratheasaponin A  Figure S1) [8][9][10]. Previously, the antiproliferative effects of dietary catechins and flavonoids from "green tea", such as compound 4 and quercetin derivatives, against HSC-2, MKN-45, and Caco-2 cells have been reported [15][16][17][18]. To identify the active principles, the inhibitory activities of abovementioned "tea flower" constituents against human gastric carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 cells were evaluated. As

Results and Discussion
2.1. Antiproliferative Activities of Constituents Isolated from "Tea Flower" against Human Gastric Carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 Cells Digestive tract carcinoma refers to a group malignancy located in the oral cavity, pharynx, larynx, esophagus, stomach, and large intestines and is the most common type of cancer worldwide [13]. In our previous studies of bioactive components from natural medicines isolated from the flowers of Bellis perennis, we observed antiproliferative activities of acylated oleanane-type triterpene saponins, perennisaponins A-T, against HSC-2, HSC-4, and MKN-45 cells [14]. The structures of perennisaponins resemble that of saponin constituents isolated from C. sinensis, and thus, similar activities were expected. Two analytical protocols have been developed with respect to nine acylated oleanane-type triterpene saponins, including chakasaponins I (1), II  Figure S1) [8][9][10]. Previously, the antiproliferative effects of dietary catechins and flavonoids from "green tea", such as compound 4 and quercetin derivatives, against HSC-2, MKN-45, and Caco-2 cells have been reported [15][16][17][18]. To identify the active principles, the inhibitory activities of abovementioned "tea flower" constituents against human gastric carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 cells were evaluated. As shown in Table 1 Table S1). The concentration dependencies of the antiproliferative activity of compounds 1-4 against HSC-2 cells are observed in a range of 3-100 µM for compounds 1, 2, and 4 and a range of 0.3-10 µM for compound 3, as shown in Figure S2.

Effects of Cell Cycle Distribution in HSC-2 Cells
Apoptosis and cell cycle dysfunction are closely associated biochemical processes, and any disturbance in cell cycle progression may lead to apoptotic cell death [19]. The effects of cell cycle distribution were analyzed to determine the mechanism associated with the growth inhibitory effect of chakasaponins I (1) and II (2), floratheasaponin A (3), and (-)-epigallocatechin 3-O-gallate (4) on HSC-2 cells [20,21]. The cell distribution in G0/G1, S, and G2/M phases shown in blue, red, and green areas, respectively, were determined after a 48 h incubation ( Figure 2). As shown in Table 2, compounds 1-3 significantly induce populations of the cell distribution in S and G2/M phases, but reduce that of the G0/G1 phase. However, compound 4 does not affect the cell cycle distribution at the effective concentration. These results imply that compounds 1-3 induced the cell cycle arrest at the G2/M phase.

Quantification of Apoptotic Cell Death Using Annexin-V Binding Assay in HSC-2 Cells
Apoptosis plays an important role in the homeostatic maintenance of the tissue by selectively eliminating excessive cells. On the other hand, the induction of apoptosis of carcinoma cells is also recognized to be useful in cancer treatment, since cytotoxic agents used in chemotherapy of leukemia and solid tumors are known to cause apoptosis in target cells. Thus, the induction of apoptosis of cancer cells may be useful in cancer treatment [22]. To determine the apoptosis-inducing effects of compounds 1-4 against HSC-2 cells, staining with annexin-V/7-aminoactinomycin D (7-AAD) as a marker of early and late apoptotic events was performed ( Figure 3) [23]. As shown in Table 3, the percentage of total apoptosis of compounds 1-3 increase in a concentration-dependent manner. However, compound 4 does not induce apoptotic cell death by this annexin-V-binding assay.

Evaluation of Apoptotic Morphological Changes in HSC-2 Cells
Representative morphological features of apoptosis in HSC-2 cells were examined under an inverted light fluorescence microscope using 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) as a staining agent [24]. As shown in Figure 4, the cell shrinkage caused by compounds 1-3 was mediated partially through apoptosis induction as nuclear chromatin condensation. Nuclei of the cells treated with compound 4 showed the obvious changes only slightly, while evident nuclear condensation was observed in cells treated with compounds 1-3.

DNA Fragmentation in HSC-2 Cells
DNA fragmentation in HSC-2 cells treated with compounds 1-4 were examined. Apoptotic cells display condensed chromatin and fragmented nuclei, but nonapoptotic cells maintain their structure [24][25][26]. As shown in Figure 5, DNA ladder formation, which is indicative of apoptosis, is observed in HSC-2 cells treated with compounds 1-3 for 48 h at effective concentrations. This fragmentation shows the same pattern as those treated with actinomycin D at a concentration of 0.1 µM.

Effects of Caspase-3/7 in HSC-2 Cells
Caspase play a central role in the apoptotic signaling. During apoptosis, caspase activity contributes to the degradation of DNA and leads to further disruption of cellular components, resulting in alterations of cell morphology [22,[27][28][29]. To confirm that caspases are involved the enzyme activity of caspase-3/7 in HSC-2 cells after coculture with compounds 1-4, the activities of caspase-3/7 were measured by Muse ® Cell Analyzer ( Figure 6). As shown in Table 4, the activation of caspase-3/7 by compounds 1-3 was found to occur in a concentration-dependent manner. These results suggest that the antiproliferative effects of acylated saponin constituents isolated from "tea flower" (1-3) against HSC-2 involve apoptotic cell death via activation of caspase-3/7. The efficacies of these saponins were found to be stronger than that of compound 4, the most abundant polyphenol constituent in "green tea".

Effects of Caspase-3/7 in HSC-2 Cells
Caspase play a central role in the apoptotic signaling. During apoptosis, caspase activity contributes to the degradation of DNA and leads to further disruption of cellular components, resulting in alterations of cell morphology [22,[27][28][29]. To confirm that caspases are involved the enzyme activity of caspase-3/7 in HSC-2 cells after coculture with compounds 1-4, the activities of caspase-3/7 were measured by Muse ® Cell Analyzer ( Figure 6). As shown in Table 4, the activation of caspase-3/7 by compounds 1-3 was found to occur in a concentration-dependent manner. These results suggest that the antiproliferative effects of acylated saponin constituents isolated from "tea flower" (1-3) against HSC-2 involve apoptotic cell death via activation of caspase-3/7. The efficacies of these saponins were found to be stronger than that of compound 4, the most abundant polyphenol constituent in "green tea".

Effects of Caspase-3/7 in HSC-2 Cells
Caspase play a central role in the apoptotic signaling. During apoptosis, caspase activity contributes to the degradation of DNA and leads to further disruption of cellular components, resulting in alterations of cell morphology [22,[27][28][29]. To confirm that caspases are involved the enzyme activity of caspase-3/7 in HSC-2 cells after coculture with compounds 1-4, the activities of caspase-3/7 were measured by Muse ® Cell Analyzer ( Figure 6). As shown in Table 4, the activation of caspase-3/7 by compounds 1-3 was found to occur in a concentration-dependent manner. These results suggest that the antiproliferative effects of acylated saponin constituents isolated from "tea flower" (1-3) against HSC-2 involve apoptotic cell death via activation of caspase-3/7. The efficacies of these saponins were found to be stronger than that of compound 4, the most abundant polyphenol constituent in "green tea".

Cell Cycle Analysis
The cell cycle distribution analysis was measured by Muse ® Cell Analyzer (Merck-Millipore, Darmstadt, Germany) using a Muse Cell Cycle Assay Kit (Merck-Millipore) according to the manufacturer's instructions. Briefly, HSC-2 cells were inoculated into a 6-well tissue culture plate (2 × 10 5 cells/1.5 mL/well) and cultured in MEM containing 10% FBS, penicillin G (100 U/mL), and streptomycin (100 µg/mL) at 37 • C under a 5% CO 2 atmosphere. After 24 h incubation, 500 µL/well of medium containing the test sample was added. After 48 h treatment, the cells were harvested by trypsinization, and suspended in 300 µL of PBS(-). The cells were added in 700 µL of ice-cold ethanol and incubated overnight at −20 • C. The fixed cells were collected by centrifugation at 300× g for 5 min and washed twice with 250 µL of PBS(-), then suspended in 200 µL of cell cycle reagent and incubated for 30 min in dark [30]. Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration: 0.5%). 5-FU was used as a reference compound.

Annexin-V/7-AAD Assay
Apoptosis was measured by a Muse Annexin-V and Dead Cell Kit (Merck-Millipore) according to the manufacturer's instructions. Briefly, HSC-2 cells were inoculated into a 6-well tissue culture plate (2 × 10 5 cells/1.5 mL/well). After 24 h incubation, 500 µL/well of medium containing the test sample was added. After 24 h treatment, the cells were harvested by trypsinization and a 100 µL cell suspension was labeled for 20 min in the dark with the same volume of Muse Annexin-V and Dead Cell Reagent. Subsequently, quantitative detection of annexin-V/7-AAD-positive cells was performed using the Muse ® Cell Analyzer. Cells stained with annexin-V only were defined as early apoptotic, while annexin-V and 7-AAD double-stained cells were defined as late apoptotic [14]. Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration: 0.5%). Camptothecin was used as a reference compound.

DAPI Staining for Morphological Analysis
HSC-2 cells (2 × 10 5 cells/2 mL/well) were seeded onto coverslips in a 6-well tissue culture plate and cultured in MEM containing 10% FBS, penicillin G (100 U/mL), and streptomycin (100 µg/mL) at 37 • C under 5% CO 2 atmosphere. After 24 h incubation, the medium was replaced with 2 mL fresh medium per well containing the test sample; then, the cells were cultured for 48 h. Next, the medium was removed and washed twice with PBS(-) and fixed with 4% paraformaldehyde phosphate buffer solution (pH 7.4, Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were permeabilized by 0.2% Triton X-100 in PBS(-), stained with DAPI (1 µg/mL in PBS(-)), and were observed by fluorescence microscopy (EVOS ® FL Cell Imaging System, Thermo Fisher Scientific, Waltham, MA, USA) [14]. Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration: 0.5%). Camptothecin was used as a reference compound.

Agarose Gel Electrophoresis for the Detection of DNA Fragmentation
HSC-2 cells were inoculated into a 6-well tissue culture plate (2 × 10 5 cells/1.5 mL/well). After 24 h incubation, 500 µL/well of medium containing the test sample was added. After 48 h treatment, the cells were harvested and suspended in a solution containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% sodium dodecyl sulfate (SDS) at room temperature for 30 min. The lysates were incubated with RNase A (100 µg/mL) for 1 h at 37 • C, then proteinase K (500 µg/mL) was added and incubated at 50 • C for 2 h. DNA was extracted twice with an equal volume of 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol and was purified by ethanol precipitation. The DNA was subjected to electrophoresis on 2.0% agarose gels and stained by ethidium bromide. The DNA bands were detected under UV illumination [24]. Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration: 0.5%). Actinomycin D was used as a reference compound.

Caspase-3/7 Assay
Apoptotic status based on caspase-3/7 activation was measured by a Muse Caspase-3/7 Kit (Merck-Millipore). Briefly, HSC-2 cells were inoculated into a 6-well tissue culture plate (2 × 10 5 cells/1.5 mL/well). After 24 h incubation, 500 µL/well of medium containing the test sample was added. After 48 h treatment, the cells were harvested by trypsinization and stained according to the manufacturer's instructions. Subsequently, quantitative detection of caspase-3/7-positive cells was performed using the Muse ® Cell Analyzer [31][32][33]. Each test compound was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration: 0.5%). Camptothecin was used as a reference compound.

Statistics
Values are expressed as means ± S.E.M. Significant differences were calculated using Dunnett's test. Probability (p) values less than 0.05 were considered significant.

Conclusions
Acylated oleanane-type triterpene saponins obtained from the flower buds of C. sinensis (tea flower), namely chakasaponins I (1) and II (2) and floratheasaponin A (3), and the most abundant polyphenol constituent in "green tea", (-)-epigallocatechin 3-O-gallate (4), showed antiproliferative activities against human digestive tract carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 cells. These activities of the saponins (1-3, IC 50 = 4.4-14.1, 6.2-18.2, 4.5-17.3, and 19.3-40.6 µM, respectively) were more potent than those of 4 (IC 50 = 28.3, 27.2, >100, and >100 µM, respectively). In our previous study, we have demonstrated that a similar acylated oleanane-type triterpene saponin obtained from the flowers of Bellis perennis (Asteraceae), perennisaponin O, showed a relatively strong activity against HSC-2, HSC-4, and MKN-45 cells (IC 50 = 11.2, 14.3, and 6.9 µM, respectively). Furthermore, the mechanism of action of perennisaponin O against HSC-2 was found to involve apoptotic cell death [14]. To clarify the mechanisms of action of compounds 1-3 against HSC-2 cells, effects of cell cycle distribution were analyzed at each effective concentration. The present study demonstrated a significant inhibition of cell proliferation by compounds 1-3 with cell cycle arrest occurring at the G2/M phase in a concentration-dependent manner. These saponins (compounds 1-3) efficiently induce apoptosis in HSC-2 cells, as demonstrated by annexin V/7-AAD assay, morphological changes, and DNA fragmentation. Furthermore, the apoptotic cell death triggered by compounds 1-3 in HCS-2 cells was dependent on the activation of caspase-3/7. The efficacy of these saponins (1-3) as apoptosis inducers was found to be higher than that of compound 4. Recently, compound 4 has received great attention in cancer research related to cancer preventive effects [34][35][36], the synergistic enhancement by the combination with different anticancer drugs [37,38], and clinical applications [39][40][41]. It is well recognized that compound 4 is able to bind to multiple molecular targets, thus, it can affect a range of signaling pathways, resulting in growth inhibition, apoptosis or suppressions of invasion, angiogenesis, and metastasis [42]. Ability of compound 4 to induce cell death in cancer cells is considered a key mechanism related to its anticancer function [43]. There are conflicting reports of apoptotic and nonapoptotic cell death induced by compound 4, and the exact molecular mechanisms have not been fully elucidated yet. However, most of the previous reports have concluded that this compound induces caspase-mediated apoptosis in various tumor cells via the mitochondrial pathway [44,45] or via the death receptor [46][47][48]. Conversely, several reports demonstrated the involvement of nonapoptotic cell death, such as caspase-independent necrosis-like cell death [49] or reactive oxygen species (ROS)-mediated lysosomal membrane permeabilization [50]. The detailed mechanism of action of the antiproliferative activity of compound 4 needs further studies. In conclusion, we herein described that the saponin constituents of "tea flower" compounds 1-3 showed some antiproliferative activities against HSC-2 cells by the apoptotic pathway via caspase-3/7 activation. On the basis of the abovementioned evidence, these saponins can potentially be useful for the treatment and/or prevention of the digestive tract cancer. Further investigations are recommended.