Oleiferasaponin C₆ from the seeds of Camellia oleifera Abel.: a novel compound inhibits proliferation through inducing cell-cycle arrest and apoptosis on human cancer cell lines in vitro

Abstract

A new oleanane-type saponin oleiferasaponin C₆ (OSC₆) was isolated and identified from Camellia oleifera seeds through NMR, HR-ESI-MS, and GC-MS spectroscopic methods. This new oleanane-type saponin exhibits potent cytotoxic activities on five human cancer cell lines (BEL-7402, BGC-823, MCF-7, HL-60 and KB), especially in HL-60 and BEL-7402 cell lines. The action mechanism of the anti-cancer activity of OSC₆ was further investigated and it showed significant induction of cell cycle arrest and apoptosis in HL-60 and BEL-7402 cell lines by down-regulating CDK4, cyclin D1 and Bcl-2; up-regulating p21, caspase-3/9, p-p53 and Bax protein expression in vitro. These results suggested that the newly isolated saponin OSC₆ is a potential therapeutic agent for the treatment of human cancer.

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Introduction

Cancer is a significant and growing cause of mortality worldwide, with an increase to 19.3 million new cancer cases per year projected for 2025.^1,2 Stomach, liver and breast cancers are three common types of cancer in most countries. In addition, oral cancer and leukemia have emerged as significant, global public health concerns.^3,4 In spite of the extensive efforts and investment in research, there remains an urgent need for development of more efficient anticancer agents with minimal side effects. Therefore, there is considerable interest in the discovery and development of novel phytochemicals, which has led to investigation of the crude plant extracts that exhibit broad-spectrum anti-tumor activity in certain tumors. Triterpenoid saponins, constitute an important group of plant secondary metabolites that show diverse pharmacological and biological activities.^5–8 Because of the growing realization that triterpenoids may be a source of medication for cancer treatment, saponins have been extracted from Camellia oleifera Abel. plant.^9–11

C. oleifera Abel. is widely cultivated throughout southern China for the edible oil within its seeds. Approximately 1 million tons of the seeds yielding 730 000 tons of defatted seeds are generated yearly in China. It should be noted that the defatted seeds contain about 10% saponins by dry weight,¹² comprising more than 30 types of saponins.^13,14 Although the seed saponins are difficult to purify and identify, recent studies of the structures and functions of saponins showed that there are 11 types of triterpenoid saponins, including 10 newly isolated types,^{10–12,15–18} and that some exhibited broad-spectrum, anti-proliferative effects at the micromole level against human lung, gastric, breast, liver and colon tumor cell lines.^10–12 However, the inhibitory mechanisms of the saponins on cancer cells have not been widely reported yet. As a part of our ongoing study of the constituents of the C. oleifera seed, we recently isolated a new oleanane-type saponin from the C. oleifera seeds that showed better anti-cancer activities compared to other six saponins we previous identified (Fig. S1†).^10,11 In the present study, we report the isolation and structural elucidation of this new saponin, namely oleiferasaponin C₆ (OSC₆), along with possible molecular mechanisms underlying its effects on multiple human cancer cell lines. We observed that OSC₆ significantly inhibited proliferation of human hepatocellular carcinoma (BEL-7402) and human promyelocytic leukemia (HL-60) cells. Moreover, OSC₆ induced cell apoptosis and led to a significant down-regulation of anti-apoptotic protein Bcl-2, up-regulation of pro-apoptotic protein Bax, and activation of the p53 pathway. Our results have provided the theoretical basis for the development of OSC₆ as a potential novel anticancer agent.

Materials and methods

General experimental procedures

UV (ultraviolet) spectra were recorded on a Hitachi U-5100 spectrophotometer. IR (infrared) spectra were measured on a Nicolet 8700 FT-IR spectrophotometer with KBr pellets. NMR spectra were obtained on an Agilent DD2 (600 MHz) spectrometer using pyridine-d₅ as solvent. High resolution electro-spray ionization mass spectrometry (HR-ESI-MS) was run on an Electrostatic Field Orbital Trap Mass Spectrometer (Thermo Scientific). Semi-preparative HPLC was performed on a Varian Prostar HPLC Model 325 instrument (Varian, Mulgrave, Australia) and a YMC-Pack ODS-A semi-preparative HPLC column (250 mm × 10 mm i.d., 5 μm, YMC Corp., Ltd., Kyoto, Japan), with further HPLC purifications performed on a Waters 2695 separation module combined with a Waters 2489 UV detector and an Agilent Zorbax Eclipse Plus C₁₈ column (250 mm × 4.6 mm i.d., 5 μm, Agilent Corp., Palo Alto, CA, USA). GC-MS analyses were conducted on a GCMS-QP2010S (Shimadzu Corp., Kyoto, Japan) with DB-5MS column (i.d. = 0.25 μm, length = 30 m, Agilent Technologies).

Plant material

The seeds of C. oleifera were collected in Huangshan, Anhui Province, P. R. China, in October 2012. The plant material was identified by one of the authors (Prof. G.H. Bao), and a voucher specimen (no. 2012-10 Hou) was deposited in State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University.

Extraction and isolation

The tea seeds (2.0 kg) were crushed into powder and extracted three times with 70% EtOH (3 × 10 L) at 60 °C under reflux for 4 h each time. The extract was subjected to reduced pressure evaporation to obtain EtOH concentrated solution (350 g), which was suspended in H₂O and extracted successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH fraction (80 g) was subjected to CC on silica gel and eluted with a gradient of EtOAc–MeOH (100 : 0, 90 : 10, 80 : 20, 70 : 30, 60 : 40, 50 : 50, 40 : 60, 30 : 70, 20 : 80, 10 : 90, 0 : 100, each 8.0 L). Each fraction was collected, yielding eight major fractions (A–H), and analyzed by TLC. Fraction G (6.5 g) was submitted to CC on Sephadex LH-20 in MeOH to remove the pigments and flavones, yielding four major fractions (I–IV). Fraction II (2.0 g) was subjected to semipreparative HPLC [YMC-Pack ODS-A, CH₃CN-0.5% aqueous HCOOH (40 : 60, v/v), 2 mL min⁻¹] to yield five fractions [Fr. 1 (0.83 g), Fr. 2 (0.12 g), Fr. 3 (0.06 g), Fr. 4 (0.11 g), and Fr. 5 (0.55 g)]. Fraction 4 was further purified by HPLC [Agilent C₁₈, CH₃CN-0.5% aqueous HCOOH (44 : 56, v/v), 1 mL min⁻¹] to afford oleiferasaponin C₆ (6.7 mg).

OSC₆: white amorphous powder; UV (MeOH) λ_max nm (log ε): 208 (3.98), 254 (4.17), 279 (4.32); IR (KBr) ν_max (cm⁻¹): 3416, 2950, 2927, 1716, 1638, 1450, 1384, 1309, 1164, 1078, 1046; ¹H NMR (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) spectroscopic data, was shown in Table 1, Fig. S3 and S4;† HR-ESI-MS (positive ion mode): m/z 1345.5824 [M + Na]⁺ (calcd for C₆₅H₉₄O₂₈ Na, 1345.5829), the data were shown in Fig. S2.†

Table 1 ¹H and ¹³C NMR spectroscopic data of oleiferasaponin C₆ in pyridine-d₅^a

Position	δC	δH	Position	δC	δH
a ^1H (δ ppm, J Hz, s: singlet; d: doublet; brs: broad singlet; m: multiplet). Ac: acetyl; Cin: cinnamoyl; GlcA: glucuronic acid; Gal: galactose; Xyl: xylose.
1	38.0	0.89m, 1.41m	22-O-Cin
2	25.1	1.84m, 2.07m	Cin-1	167.1
3	84.5	4.01m	Cin-2	119.2	6.57d (15.6)
4	55.0		Cin-3	144.6	7.93d (16.2)
5	48.3	1.39m	1′	134.8
6	20.3	0.95m, 1.35m	2′, 6′	128.3	7.31m
7	32.3	1.14m, 1.50m	3′, 5′	129.1	7.29m
8	40.2		4′	130.5	7.30m
9	46.6	1.75m	3-O-
10	35.9		GlcA-1′	104.0	4.75d (7.2)
11	23.7	1.76m, 1.85m	GlcA-2′	77.8	4.48m
12	123.1	5.39brs	GlcA-3′	84.6	4.27m
13	142.8		GlcA-4′	69.5	4.49m
14	41.6		GlcA-5′	77.0	4.10m
15	34.5	1.59m, 1.81m	GlcA-6′	169.9
16	68.1	4.52brs	COOMe	52.1	3.69s
17	48.2		Gal-1′′	102.9	5.87d (7.8)
18	39.9	3.13m	Gal-2′′	73.6	4.47m
19	47.1	1.42m, 3.09m	Gal-3′′	75.3	4.39m
20	36.2		Gal-4′′	70.4	4.31m
21	79.4	6.64d (10.2)	Gal-5′′	76.4	4.48m
22	74.2	6.40d (10.2)	Gal-6′′	62.2	4.50 (2H, m)
23	210.1	9.93s	Gal-1′′′	101.6	5.68d (7.2)
24	11.0	1.45s	Gal-2′′′	83.9	4.52m
25	15.7	0.79s	Gal-3′′′	75.0	4.29m
26	16.7	0.80s	Gal-4′′′	70.4	4.51m
27	27.3	1.78s	Gal-5′′′	76.2	4.31m
28	63.3	3.42m, 3.63m	Gal-6′′′	61.6	4.34 (2H, m)
29	29.3	1.09s	Xyl-1′′′′	107.7	5.03d (7.8)
30	20.0	1.33s	Xyl-2′′′′	76.3	4.21m
21-O-Ac			Xyl-3′′′′	78.2	4.05m
Ac-1	170.9		Xyl-4′′′′	70.7	4.33m
Ac-2	20.1	2.08s	Xyl-5′′′′	67.6	3.48m, 4.43m

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Acid hydrolysis and GC-MS analysis of the sugar moieties in OSC₆

The configuration of sugar units was established after hydrolysis of OSC₆ with 1 M HCl, pyridine containing L-cysteine methyl ester hydrochloride, trimethylsilylimidazole and determination of the retention times by GC-MS operating under the experimental conditions previously reported by Zong et al.¹⁰ The standard sugar samples were subjected to the same reaction and GC-MS conditions. The sugar units of OSC₆ were identified by comparison with authentic samples: D-xylose (t_R 16.72 min), D-galactose (t_R 22.30 min), D-glucuronic acid methyl ester (t_R 23.34 min). D-Xylose, D-galactose and D-glucuronic acid methyl ester were identified in a ratio of 1 : 2 : 1 for OSC₆.

Cell culture

Human hepatocellular carcinoma BEL-7402, human gastric carcinoma BGC-823, human breast cancer MCF-7, human promyelocytic leukemia HL-60 and human oral epidermoid carcinoma KB cell lines were obtained from KeyGEN BioTECH (Nanjing, China). Cells were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin (100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin) and 2 mM L-glutamine at 37 °C in a 5% CO₂ humidified atmosphere.

Cell viability assay

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described Zong et al.¹¹ Briefly, cell lines in culture medium were placed in a cell of a 96-well plate at a concentration of 4 × 10³ cells per mL and incubated at 37 °C in 5% CO₂ for 24 h. After 24 h, an additional 100 μL of complete medium with either: no additions (negative control), 0.1% DMSO (solvent control), 10 μg mL⁻¹ Taxol (positive control), or different concentrations of OSC₆ ranging from 0.39 μM to 50 μM were added, and incubated for 72 h. Then, 20 μL of MTT solution (5 mg mL⁻¹) were added to the culture medium, and incubated for 4 h. Next, the medium was removed, and the formazan was dissolved with 150 μL DMSO. Absorbance values were measured at 490 nm using an enzyme-linked immunosorbent Reader (EL-x800, BioTek Instruments, Winooski, VT, USA). The IC₅₀ value was the concentration of OSC₆ that resulted in 50% inhibition of cell growth, which was graphically calculated as a comparison with control growth.

Cell cycle analysis

HL-60 and BEL-7402 cells were seeded in 6-well plates at 5 × 10⁵ cells per well, and incubated for 24 h until adherent. Next, HL-60 and BEL-7402 cells were treated with various concentrations of OSC₆ for 72 h, respectively. After treatment with OSC₆, the cells were harvested, washed, suspended in ice-cold PBS, and fixed in 70% ethanol at 4 °C overnight. The cells were stained for total DNA content with RNase A and propidium iodide (PI) staining buffer according to the manufacturer's instructions (Becton Dickinson). Cell cycle distribution was analyzed using a flow cytometer (Becton Dickinson, San Jose, CA, USA) and ModFit software V3.0 (Verity Software House, Topsham, ME, USA).

Cell apoptosis analysis

Cell apoptosis was assessed using the Annexin V-APC/7-AAD double-staining apoptosis detection kit (Becton Dickinson, San Jose, CA, USA). In brief, HL-60 and BEL-7402 cells were treated with DMSO or OSC₆ for 24 h. Next, cells were washed with ice-cold PBS, and incubated for 15 min at room temperature in the dark. The cells were collected and stained according to the manufacturer's instructions. The apoptosis data acquisition and analysis was performed by a FACS Calibur flow cytometer. Basal apoptosis were determined on negative control cells.

Western blot analysis

HL-60 and BEL-7402 cells (2 × 10⁵ cells per well) were seeded in 6-well plates. After 24 h, the medium was replaced with fresh culture medium containing different concentrations of OSC₆ or DMSO for 72 h. Then cultured cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors (Pierce, Rockford, IL, USA). Samples were maintained on ice for 30 min and then centrifuged at 13 000 × g for 10 min, and the supernatants were collected and quantified. Total protein concentration was quantified using BCA protein assay. Equal amount of samples were subjected to 12% SDS-PAGE, transferred to nitrocellulose membranes, which was blocked for 1 h at room temperature using 5% non-fat dry milk dissolved in PBS containing 0.1% Tween-20 (PBS-T). The membranes were washed 3 times with PBS-T, and then incubated overnight with the primary antibodies for one of the following: CDK4, cyclin D1, p21, p-p53, p53, Bax, Bcl-2, caspase-3, caspase-9, cytochrome c, and β-actin (KeyGEN BioTECH). After washing with PBS-T, the membranes were incubated with goat anti-rabbit IgG or with goat anti-mouse IgG HRP-conjugated secondary antibodies for 2 h. The immunoreactive bands were detected using an enhanced chemiluminescence (ECL) detection system (Bio-Rad). The protein bands were analyzed using ChemiDoc XRS⁺ system equipped with Image Lab software v.4.1 (Bio-Rad).

Statistical analysis

All activity data are presented as mean ± SD from at least three independent experiments. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison using GraphPad software (Prism, San Diego, CA, USA). A P value of <0.05 was statistically significant.

Results and discussion

Isolation and characterization of the triterpenoid saponin

The n-BuOH fraction obtained from the 70% EtOH extract of defatted C. oleifera seeds was subjected to repeated column chromatography (silica gel, Sephadex LH-20), and HPLC to afford a new oleanane-type saponin (Fig. S9†). The structure was established mainly by NMR experiments and HR-ESI-MS.

OSC₆ was separated as a white amorphous powder. The molecular formula C₆₅H₉₄O₂₈ was deduced from the HR-ESI-MS [M + Na]⁺ ion at m/z 1345.5824. The IR spectrum (cm⁻¹) showed the presence of hydroxyl (broad peak around 3416), carbonyl (1716), olefinic (1638), and ether (1078, 1046) functional groups. The ¹H and ¹³C NMR spectroscopic data (Table 1) for triterpenoid aglycone moieties of the compound were similar to those reported for isotheasaponin B₂,¹⁹ except for the location of a C-23 group. The presence of signals at δ_C 210.1 and δ_H 9.93, indicated that the replacement of the 23-CH₃ group in isotheasaponin B₂ by the 23-CHO group (Fig. 1). In addition, the HMBC (heteronuclear multiple-bond correlation) correlations (Fig. 1) between δ_H 6.64 (H-21 of the aglycone), 2.08 (H-2 of acetyl group) and δ_C 170.9 (C-1 of acetyl group), as well as between δ_H 6.40 (H-22 of the aglycone) and δ_C 167.1 (C-1 of cinnamoyl group), indicated that the acetyl and cinnamoyl groups were located at C-21 and C-22 of the triterpenoid aglycone, respectively. Furthermore, the NOESY (nuclear Overhauser effect spectroscopy) correlations (Fig. 1) between H-21 at δ_H 6.64 and H-29 at δ_H 1.09, as well as those between H-22 at δ_H 6.40 and H-30 at δ_H 1.33, suggested that H-21 and H-22 are in α- and β-orientations, which means that the acetyl group at C-21 and the cinnamoyl group at C-22 are β- and α-orientations, separately. Thus, the aglycone of OSC₆ was elucidated as 16α-hydroxy-21β-O-acetyl-22α-O-cinnamoyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene, which was identified to be a new aglycone. In addition, the NMR spectroscopic data of the sugar units (Table 1) were very similar to the sugar units of oleiferoside D.⁹ Moreover, the location of the glycosidic chain was confirmed by the ¹H–¹H COSY (correlation spectroscopy), HSQC (heteronuclear single-quantum correlation), HMBC and NOESY experiments (Fig. 1, S5–S8†). The identifications of the sugar units were further confirmed by acid hydrolysis and GC-MS analysis, which revealed one unit of D-glucuronic acid methyl ester (GlcA), two units of D-galactose (Gal) and one unit of D-xylose (Xyl). On the basis of the above analysis, all the proton and carbon signals due to the aglycone and glycosidic portion were unambiguously assigned by 1D- and 2D-NMR experiments. The chemical structure of OSC₆ was established to be 16α-hydroxy-21β-O-acetyl-22α-O-cinnamoyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3β-O-[β-D-galactopyranosyl-(1 → 2)]-[β-D-xylopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3)]-β-D-glucopyranosiduronic acid methyl ester.

Fig. 1 Structure and key HMBC, NOESY correlations of OSC₆. (A) Structure of OSC₆. (B) Key HMBC and NOESY correlations of OSC₆.

OSC₆ inhibits the viability of five human tumor cell lines

The effect of the isolated compound on five human cancer cell lines (BEL-7402, BGC-823, MCF-7, HL-60 and KB) was tested using the MTT assay. The saponin OSC₆ exhibited potent anti-proliferative activities, with IC₅₀ values of 4.023 μM (BEL-7402), 6.001 μM (BGC-823), 9.016 μM (MCF-7), 1.876 μM (HL-60) and 6.119 μM (KB). OSC₆ was especially potent toward HL-60 and BEL-7402 cells (Fig. S10 and Table S1†). The cytotoxicity of OSC₆ was also measured in normal liver L-O2 cell line. There was a 5-fold higher in normal liver L-O2 cell (IC₅₀ = 19.973 μM) than liver cancer BEL-7402 cell line.

Structure–activity relationships were inferred by comparison of the structures and anti-proliferative activities of the seven types of saponins and total saponins isolated from C. oleifera with known cytotoxic activity.^10,11 Among these compounds, OSC₆ is the most potent against HL-60 and BEL-7402 cells (Table S2†). The cinnamoyl group at C-22 plays an important role in the anti-proliferative activity of oleanane-type saponins.^12,20,21 Furthermore, in comparison with that of oleiferasaponin C₃,¹⁰ it seems that the cinnamoyl group at position C-22, rather than C-28, and the free hydroxy group at C-28 in OSC₆ may also play important roles in cytotoxicity. Thus, the activity of OSC₆ depends not only on an isolated structural factor, but also on a combination of properties at both the aglycone and the sugar moieties.²²

OSC₆ anti-proliferative activities: induction of cancer cell apoptosis

Both HL-60 and BEL-7402 cells were treated with OSC₆ followed by staining for apoptosis with Annexin V and APC/7-AAD staining (Fig. 2). OSC₆ treatments resulted in an increased number of apoptotic cells. After 72 h of treatment with OSC₆ (1 μM and 2 μM), there was an increase in the percentage of apoptotic HL-60 cells (17.27 and 60.72%, respectively) compared with the percentage of apoptotic cells in the negative control (4.87%). The effect on BEL-7402 was similar to that on HL-60 cells.

Fig. 2 OSC₆ induces apoptosis in HL-60 and BEL-7402 cells as determined by flow cytometry. (A) HL-60 and BEL-7402 cells were treated with OSC₆ for 72 h with indicated concentrations. Lower right (LR) quadrant represents percentage of early apoptotic cells, upper right (UR) quadrant represents percentage of late apoptotic cells. (B) The percent of apoptotic HL-60 cells when treated with OSC₆ with indicated concentrations. (C) The percent of apoptotic BEL-7402 cells when treated with OSC₆ with indicated concentrations.

Mitochondrial cytochrome c (Cyto-C) release and caspase activation are important hallmarks of apoptosis.²³ When cytosolic Cyto-C meets procaspase-9, the apoptosome is formed, which subsequently stimulates proteolytic activation of caspase-3.²⁴ Protein levels of Cyto-C, caspase-3 and caspase-9 were measured using western blotting (Fig. 3). OSC₆ treatment resulted in increased accumulation of Cyto-C and formation of the caspase-3 and caspase-9 active forms (Fig. 3F–H). To further investigate the mechanism underlying OSC₆-induced apoptosis, we examined the levels of anti-apoptotic and pro-apoptotic proteins. The action of Bax is counteracted by an anti-apoptotic Bcl-2 family member, such as Bcl-2, which can inhibit mitochondrial pro-apoptotic events.²⁵ Both HL-60 and BEL-7402 cells were treated with OSC₆ (0, 1, 1.5 and 2 μM) for 72 h followed by western blotting analysis of protein expression. We observed increased Bax pro-apoptotic protein expression (Fig. 3C) and decreased Bcl-2 anti-apoptotic protein expression (Fig. 3D) following OSC₆ treatment. Inhibition of anti-apoptotic proteins of the Bcl-2 family members can also result in activation of the mitochondrial apoptotic signaling pathway.²⁶ Apoptosis is also regulated by the balance between Bcl-2 and Bax proteins. Analysis of signal intensity confirmed that the Bax/Bcl-2 ratio decreased in a dose-dependent manner (Fig. 3E). These results indicated that activation of apoptotic pathways is a mechanism underlying OSC₆-induced cell death.

Fig. 3 OSC₆ treatment induces changes in apoptosis-related protein expression. HL-60 and BEL-7402 cells were treated with OSC₆ for 72 h with indicated concentrations. (A) and (B) Expression of precursor and active forms of Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. β-Actin was used as internal control. (C), (D), (F), (G), (H) Apoptosis in each treatment is significantly different with control. (E) Analysis of signal intensity confirmed that the Bax/Bcl-2 ratio decreased in a dose-dependent manner. Each value is expressed as the mean ± SD (n = 3), compared to the control (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, non-significant).

OSC₆ arrested cells in the G0/G1 phases of the cell cycle

Distribution of the cells between the different phases of the cell cycle was analyzed by flow cytometry of PI stained cells after a 72 h exposure to OSC₆. OSC₆ clearly perturbed cell cycle progression, inducing an increase in the G0/G1 fraction and a decrease in the S and G2/M fraction (Fig. 4A), reflecting the arrest at G0/G1 in HL-60 cells. Exposure of BEL-7402 cells to OSC₆ for 72 h also led to an accumulation of cells in the G0/G1 phase, with a corresponding decrease of cells in the S and G2/M phases. Thus, the inhibition of cell proliferation provoked by a 72 h exposure to OSC₆ is mediated by cell cycle arrest in the G0/G1 phase.

Fig. 4 OSC₆ treatment induces G1 cell-cycle arrest in HL-60 and BEL-7402 cells. (A) HL-60 and BEL-7402 cells were treated with OSC₆ for 72 h with indicated concentrations, stained with PI and analyzed for cell-cycle phase using flow cytometry. The distributions (percentage) of cells in different phases of the cell-cycle (G0/G1, S, and G2/M) were determined after treatment with OSC₆. (B) Cell-cycle regulatory proteins CDK4 and cyclin D1 with β-actin as internal control were examined using western blot analyses. (C) and (D) The results of the western blotting in CDK4 and cyclin D1 were evaluated in three independent experiments and normalized with β-actin. Compared to the control (*** P < 0.001, ns, non-significant).

Cyclin-dependent kinase 4 (CDK4) is a serine/threonine kinase that forms heterodimers with D-type cyclins (cyclin D) and is one of the central promoters of the G1-S transition of the cell cycle.²⁷ We analyzed the expression of the cell cycle promoters CDK4 and cyclin D1 (Fig. 4B–D). Western blot analysis revealed that the OSC₆-mediated G0/G1 arrest in HL-60 and BEL-7402 cells was accompanied by the downregulation of CDK4 and cyclin D1. Furthermore, levels of p21 were upregulated after OSC₆ treatment (Fig. 5A and B). Cell cycle progression is facilitated by cyclins and CDKs and suppressed by CDK inhibitors p21 and p53. Consistent with the observed G0/G1 cell cycle arrest, exposure to OSC₆ diminished the expression of CDK4 and cyclin D1 in both HL-60 and BEL-7402 cells.

Fig. 5 OSC₆ treatment activates p53 pathway in cancer cell lines. (A) HL-60 and BEL-7402 cells were treated with OSC₆ for 72 h with indicated concentrations. The protein levels of p53, phosphorylated p53, and p21 were detected by western blotting analysis, β-actin was used as internal control. (B), (C) and (D) The results of the western blotting in p-p53, p53 and p21 were evaluated in three independent experiments and normalized with β-actin. Compared to the control (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, non-significant).

OSC₆ treatment activates p53 pathway of tumor cell lines

p53 can be activated by many intrinsic and extrinsic factors, and its activation is critical for cell fate determination. Activation of p53 can induce cell apoptosis and bring about cell cycle arrest.²⁸ In addition, p53 regulates numerous downstream molecules, several of which are involved in the death receptor- and mitochondria-mediated apoptotic pathways, which plays a key role in drug-based induction of apoptosis.^29,30 Consistent with these findings, we observed a significant p53 accumulation and increase in the population of phosphorylated p53 (p-p53) following OSC₆ treatment (Fig. 5). We also detected increased expression of the p53-target protein p21 (Fig. 5). This suggests that p53 plays a vital role in OSC₆-induced cell cycle arrest and apoptosis.

Conclusions

In summary, the present study reveals that the anti-cancer activity of the novel triterpenoid saponin OSC₆ involves activation of the p53 pathway, responsible for inducing apoptosis and G0/G1 cell cycle arrest in cancer cells. OSC₆, as a natural product isolated from the C. oleifera seeds, exhibits potent anti-proliferative abilities against human liver, gastric, breast, leukemia and oral epidermoid cancer cells in vitro. An important finding in this study was that OSC₆ induced G0/G1 cell cycle arrest that was associated with the up-regulation of p21 and down-regulation of CDK4 and cyclin D1. Moreover, OSC₆ treatment induced apoptosis, signaled through a change in the ratio of Bax/Bcl-2, cytochrome c release, caspase-3 and -9 activation, and p53. In the future, efforts should be focused on saponin's anticancer effect in animal models, and its mechanism pathway should be investigated in more detail.

Acknowledgements

This research was supported by the projects “Nutrition and Quality & Safety of Agricultural Products, Universities Leading Talent Team of Anhui Province”; The Earmarked Fund for Modern Agro-industry Technology Research System in Tea Industry of Chinese Ministry of Agriculture (nycytx-26) and Natural Science Foundation for Distinguished Young Scholars of Anhui Province (1608085J08).

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Footnote

† Electronic supplementary information (ESI) available: HR-ESR-MS, NMR spectra and anti-proliferative activity data of OSC₆. See DOI: 10.1039/c6ra14467e

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