Antitumour Effect of Sesquiterpene (+)-chabranol on Four Human Cancer Cell Lines by Inducing Apoptosis and Autophagy

Abstract

Objective:

To investigate the effects and mechanisms of sesquiterpene (+)-chabranol on proliferation of a panel of four human tumour cell lines (BGC-823, SGC-7901, SSMC-7721 and HepG2).

Methods:

Cell viability was assessed using a standard methyltetrazolium assay; cell-cycle analysis of BGC-823 cells was performed by flow cytometry. Transmission electron microscopy (TEM) was used to examine the ultrastructure of BGC-823 cells exposed to (+)-chabranol. Apoptosis was investigated by evaluating DNA laddering, using gel electrophoresis.

Results:

(+)-Chabranol had a marked time- and concentration-dependent inhibitory effect on BGC-823 cell proliferation. The effect was less marked in SGC-7901, SSMC-7721 and HepG2 cells. Exposure of BGC-823 cells to (+)-chabranol arrested the cell cycle at G1. Evidence of apoptosis and autophagy was observed by TEM; DNA laddering in BGC-823 cells supported the presence of apoptosis.

Conclusions:

This study suggested that (+)-chabranol has antitumour activity against BGC-823 cells, and may exert its action by inhibition of proliferation and induction of apoptosis and autophagy. With further development, (+)-chabranol may represent a potential novel treatment for poorly differentiated gastric cancer.

Keywords

(+)-CHABRANOL SESQUITERPENE BGC-823 CELLS ANTITUMOUR APOPTOSIS AUTOPHAGY

Introduction

Soft coral is a rich source of structurally novel sesquiterpenes.^1–7 Sesquiterpenes have a wide range of biological functions including antibacterial actions,^8,9 anti-inflammatory effects^8,10–13 and cytotoxic antitumour properties,^9,13–15 in addition to a having a therapeutic effect in liver disease.¹⁶ The naturally occurring sesquiterpene, chabranol, has been under investigation for over a decade. Isolation of small quantities of (+)-chabranol from the soft coral Nephthea chabroli at the National Sun Yat-Sen University was followed by the development of a synthetic form of (+)-chabranol.^17,18 As a consequence of its limited natural supply, novel structural features and potent biological activity, (+)-chabranol has become an attractive target for synthetic drug development. The biological activity of (+)-chabranol is thought to be closely related to its chemical structure, which comprises a cyclopentane and tetrahydrofuran (THF) ring fused at C3–C5, together with two quaternary carbon atoms, a 2°hydroxyl group, and angled stereocentres concentrated within the THF ring (Fig. 1).

FIGURE 1:

Chemical structure of the sesquiterpene, (+)-chabranol (C₁₄H₂₄O₃; molecular weight: 240)¹⁷

Hepatocellular carcinoma (HCC) is associated with a poor prognosis and there are few systemic treatment options available. It is estimated that HCC is the third leading cause of cancer-related death worldwide, with mortality rates of ∼ 80% within 1 year of diagnosis.¹⁹ Gastric cancer is the second most common malignancy worldwide. It is estimated that 900 000 patients are diagnosed with gastric cancer every year, with 42% of all cases occuring in China.^20,21 Current treatment modalities for HCC and gastric cancer include surgery, radiotherapy and cytotoxic chemotherapy. There is a need to find new and effective treatment modalities, particularly pharmacological agents with low toxicity.

The present study investigated the biological activity and antitumour effect of (+)-chabranol in four human HCC and gastric cancer cell lines, and in human umbilical vein endothelial cells (HUVECs). Cell viability, proliferation and cell-cycle analysis were assessed using standard assays. Evidence of apoptosis and autophagy was also determined in BGC-823 cells, in order to provide a pathophysiological basis for a possible clinical antitumour effect of (+)-chabranol.

Materials and methods

Reagents and Cell Lines

Chabranol with a purity of over 99% was provided by Lanzhou University, Lanzhou, China. It was dissolved in sterile dimethyl sulphoxide (DMSO) at a stock concentration of 40 mg/ml, stored at –20 °C in the dark, then diluted in RPMI-1640 medium to obtain the desired concentration.

A poorly differentiated human gastric cancer cell line (BGC-823), a moderately differentiated human gastric cancer cell line (SGC-7901), a human hepatoma cell line (SSMC-7721), a human hepatoma G2 cell line (HepG2) and HUVECs were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Science, Shanghai, China. Cells were cultured at 37 °C with RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU/ml of penicillin and 100 μg/ml of streptomycin (pH 7.2) in 5% CO₂. Metabolically active cells in the mid-log phase were used in experiments.

MTT Cell Proliferation Inhibition Test

Cell viability was assessed using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay of the type widely used to measure cell proliferation and screen for anticancer drugs.^22,23 Assays were performed in triplicate. BGC-823, SGC-7901, SSMC-7721, HepG2, and HUVEC cells were seeded in 96-well flat plates at a density of 3 × 10⁴ cells/ml/well, and allowed to adhere overnight under the culture conditions described above. They were then treated with (+)-chabranol at final concentrations of 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg/ml for 24, 48 and 72 h under the culture conditions described above. Untreated cells served as a control group and a zero-adjustment well was also included. Cells were then exposed to MTT 18 μl (5 mg/ml) for 4 h, after which the supernatant was removed, 200 μl DMSO was added, and the suspension was shaken for 10 min. Absorbance per well was measured spectrophotometrically at 490 nm. Inhibition of cell proliferation was calculated by the formula: (1 – experimental group absorbance/blank control group absorbance) × 100%. The half-maximal inhibitory concentration (IC₅₀) was calculated according to the modified Karber's method.²⁴ The effect of increasing concentrations of (+)-chabranol on the various cell lines was also examined by light microscopy.

Cell-Cycle Analysis

The BGC-823 cells (3 × 10⁴ cells/ml/well) were treated with (+)-chabranol at 4.5 μg/ml for 48, then both floating and attached cells were collected and centrifuged at 157 g for 5 min before being washed twice with cold 0.01 M phosphate-buffered saline, pH 7.4. Cells were then fixed with cold 70% ethanol and stored overnight at 4 °C. A fluorochrome solution consisting of 50 μg/ml propidium iodide and 20 μg/ml RNase A was added, and cell-cycle analysis was performed with an EPICS XL™ flow cytometer (Beckman Coulter, Brea, CA, USA). All experiments were repeated three times.

Observation of Autophagy and Apoptosis by Tem

The BGC-823 cells (3 × 10⁴ cells/ml/ per well) were treated with (+)-chabranol at 4.5 μg/ml for 48 h, then both floating and attached cells were collected and centrifuged at 157 g for 5 min before being fixed overnight with 3% glutaraldehyde in 0.01 M PBS, pH 7.4 and immobilized with 10 g/l osmium tetroxide. Cells were washed after fixation and immobilization in 0.01 M PBS, pH 7.4. Graded ethanol was used to dehydrate the cells, after which they were embedded in epoxy resin. Resin blocks were cut into ultrathin sections and stained with uranyl acetate and lead citrate. Autophagic and apoptotic cell ultrastructure was examined by transmission electron microscopy (TEM) using a JEOL-1230 transmission electron microscope (JEOL, Tokyo, Japan).

Analysis of Apoptotic DNA from BGC-823 Cells

The DNA was extracted from untreated BGC-823 cells or BGC-823 cells (1 – 10 × 10⁶ cells/ ml/well) treated with 4.5 μg /ml (+)-chabranol for 24, 48 or 72 h, using a DNA Ladder Extraction Kit (DP2401; BioTeke Corporation, Beijing, China) with spin column, according to the manufacturer's instructions. For detection of apoptosis, DNA samples were loaded onto a 1% agarose gel and run at 100 V for 1 h, then visualized using ethidium bromide staining and ultraviolet light.

Statistical Analyses

Statistical analyses were performed using SPSS^® statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. Data were presented as mean ± SD. Multifactorial (balanced data) two-factor analysis of variance was used to compare statistical differences between concentrations of (+)-chabranol and exposure times. A P- value of < 0.05 was considered to be statistically significant.

Results

Compared with untreated controls, exposure to increasing concentrations of (+)-chabranol led to marked reductions in BGC-823 cell density that were associated with cell shrinkage, rounding and debris (Fig. 2). BGC-823 cell proliferation was significantly inhibited by (+)-chabranol in a concentration- and time-dependent manner (P < 0.05; Table 1). The IC₅₀ was dependent on exposure time, and was 54.81 μg/ml at 24 h, 9.05 μg/ml at 48 h and 5.49 μg/ml at 72 h. Proliferation was moderately inhibited by exposure to (+)-chabranol for 24, 48 and 72 h in SGC-7901, SSMC-7721 and HepG2 cell lines (Table 2); no statistical analyses were performed on these data. Only a small effect on proliferation of HUVECs was observed following treatment with (+)-chabranol (Table 2): as the IC₅₀ for HUVECs was approximately three times higher than that for other cell lines, a reduction from 2861.43 at 24 h to 156.23 at 72 h was considered to be small.

FIGURE 2:

Photomicrographs showing the effect of increasing concentrations of the sesquiterpene (+)-chabranol on the density of human gastric BGC-823 cells. (A) Control (untreated) BGC-823 cells were densely arrayed; (B) BGC-823 cell density was markedly reduced by high concentrations of (+)-chabranol (> 100 μg/ml); (C) intermediate concentrations of (+)-chabranol (6.25 to 100 μg/ml) resulted in BGC-823 cell density comparable with that observed at concentrations > 100 μg/ml; (D) Low concentrations of (+)-chabranol (< 6.25 μg/ml) reduced cell density relative to controls, but the effect was less marked than that seen with concentrations > 6.25 μg/ml

TABLE 1:

Inhibitory effects of increasing concentrations and exposure times to (+)-chabranol on proliferation of the poorly differentiated human gastric cancer cell line BGC-823, as determined by the methyltetrazolium assay

(+)-Chabranol μg/ml	Absorbance^a		Inhibition rate, %^b
(+)-Chabranol μg/ml	24 h	48 h	72 h	24 h	48 h	72 h
0 (control)	0.377 ± 0.027	0.678 ± 0.015	0.874 ± 0.049	0	0	0
0.78125	0.376 ± 0.032	0.428 ± 0.167^c	0.484 ± 0.004^c	11	37	45
1.5625	0.359 ± 0.035	0.380 ± 0.012^c	0.481 ± 0.014^c	12	44	45
3.125	0.357 ± 0.036	0.375 ± 0.063^c	0.445 ± 0.009^c	13	45	49
6.25	0.355 ± 0.041	0.357 ± 0.009^c	0.361 ± 0.020^c	16	47	59
12.5	0.335 ± 0.026^c	0.329 ± 0.022^c	0.330 ± 0.014^c	21	51	62
25	0.307 ± 0.036^c	0.301 ± 0.013^c	0.319 ± 0.018^c	22	54	64
50	0.288 ± 0.012^c	0.285 ± 0.024^c	0.290 ± 0.059^c	41	58	67
100	0.276 ± 0.021^c	0.193 ± 0.009^c	0.169 ± 0.028^c	52	72	81
200	0.180 ± 0.012^c	0.113 ± 0.013^c	0.092 ± 0.005^c	57	83	89

Data presented as mean ± SD of three experiments.

Absorbance per well measured at 490 nm.

Inhibitory rate calculated using the formula (1 – experimental group absorbance/blank control group absorbance) × 100%.

P < 0.05 versus the control group (multifactorial two-factor analysis of variance).

TABLE 2:

Inhibitory effect of increasing exposure times of (+)-chabranol on proliferation of human gastric (SGC-7901) and hepatoma (SSMC-7721 and HepG2) cell lines, and human umbilical vein endothelial cells (HUVECs; controls), as determined by the methyltetrazolium assay

	IC₅₀, μg/ml
Exposure time, h	SGC-7901	SSMC-7721	HepG2	HUVECs
24	116.77	103.86	67.19	2861.43
48	74.38	51.96	28.85	984.92
72	30.73	29.99	25.92	156.23

IC₅₀, half-maximal inhibitory concentration calculated using the modified Karber's method.²⁴

There was a significant decrease in the mean ± SD percentage of BGC-823 cells in S phase, from 40.65 ± 4.53% to 30.15 ± 5.93% (P < 0.05), following exposure to 4.5 μg/ml (+)-chabranol (Fig. 3). The proportion of cells in phases G₀/G₁ or G₂/M increased slightly in the presence of 4.5 μg/ml (+)-chabranol, from 58.26 ± 3.79% to 65.83 ± 5.57% and from 1.10 ± 1.56% to 4.35 ± 3.17% respectively, but this difference was not statistically significant (Fig. 3).

FIGURE 3:

Cell-cycle distribution in human gastric BGC-823 cells exposed to (+)-chabranol for 48 h. (A) Proportion of BGC-823 cells in G₀/G₁, S or G₂/M phase in the absence (controls) or presence of 4.5 μg/ml (+)-chabranol; exposure to (+)-chabranol increased the proportion of cells in G₀/G₁ phase and decreased the number of cells in S phase. Data presented as the mean ± SD of three experiments. ^aP < 0.05 (multifactorial two-factor analysis of variance). (B) Representative flow cytometry histograms demonstrating the number of BGC-823 cells in the absence and presence of 4.5 μg/ml (+)-chabranol

A proliferation of autophagic vacuoles, lipid droplets, heterochromatin and myeloid bodies was observed in BGC-823 cells exposed to 4.5 μg/ml (+)-chabranol (Fig. 4). There was also evidence of an autophagosome surrounding the mitochondria of these cells, and the rough endoplasmic reticulum was clearly visible. The autophagosome contained a cytoplasmic-like substance with fragments of injured mitochondria or endoplasmic reticulum. A large number of autophagy vacuoles were filled with lipid droplets, engulfed cellular organelles and endoplasmic reticulum, although there was no evidence of membrane structures being digested. Evidence of early apoptosis phenomena was observed as marked increases in heterochromatin and myeloid bodies.

FIGURE 4:

Subcellular structure of human gastric BGC-823 cells following exposure to 4.5 μg/ml (+)-chabranol for 48 h as visualized by transmission electron microscopy (TEM). (A) Control (untreated) BGC-823 cells; (B) control (untreated) BGC-823 cell organelles; (C) BGC-823 cells treated with (+)-chabranol, showing autophagic vacuoles (arrow) in the early stages of apoptosis; (D) organelles of BGC-823 cells treated with (+)-chabranol showing engulfed mitochondria, lipid droplets, phagocytic endoplasmic reticulum and a complete membrane structure. Increased numbers of myeloid bodies and heterochromatin were observed in early apoptosis (arrows). (E) Autophagy in which mitochondrial organelles digest themselves or other organelles (arrow), resulting in autophagic vacuoles and increased myeloid bodies

No evidence for DNA fragmentation was observed in control BGC-823 cells (Fig. 5A), whereas distinctive DNA laddering (which is indicative of apoptosis) was observed in BGC-823 cells exposed to 4.5 μg/ml (+)-chabranol (Fig. 5B). The extent of DNA fragmentation in apoptotic cells was dependent on exposure time, with a greater effect observed with longer incubation periods (i.e., 48 h or 72 h).

FIGURE 5:

Isolation of apoptotic DNA from BGC-823 cells exposed to (+)-chabranol as visualized by gel electrophoresis. (A) DNA from control (untreated) BGC-823 cells demonstrating bright, clear and uniform strips; (B) DNA from BGC-823 cells exposed to 4.5 μg/ml (+)-chabranol for 24 h (lane 1), 48 h (lane 2) or 72 h (lane 3). DNA changes after 24-h exposure were indistinct; apoptotic DNA ladders were extracted from cells following 48-h and 72-h exposures. M, marker; bp, base pairs

Discussion

Sesquiterpenes are active constituents that are found in a variety of plants used in traditional Chinese medicine. The characterization of the antitumour activity and mechanism of action of these naturally-occurring compounds has been the subject of extensive research.²⁵ In the present study, (+)-chabranol was found to have significant time- and concentration-dependent antiproliferative properties in the poorly differentiated human gastric cancer cell line, BGC-823. A moderate antiproliferative effect of (+)-chabranol was also observed in the moderately differentiated human gastric cancer cell line SGC-7901 and in the human hepatoma cell lines SSMC-7721 and HepG2, but (+)-chabranol had little effect on HUVECs. In a previous study, the cytotoxic activities of sesquiterpene lactones found in Ajania przewalskii were investigated; they were found to have very weak activity in BGC-823 cells (IC₅₀ > 100 μg/ml) and no activity in HepG2 cells.²⁶ Sesquiterpenes isolated from the red alga Laurencia tristicha have also been reported to be inactive against BGC-823 cells (IC₅₀ > 10 μg/ml).²⁷ Another study indicated that a new sesquiterpene from the fruits of Daucus carota also displayed weak cytotoxic activity against BGC-823, with an inhibition rate of 13.3%.²⁸

We hypothesized that (+)-chabranol may disrupt DNA synthesis and reduce mitotic activity in BGC-823 cells, both of which would result in cell differentiation, thereby promoting cell-cycle arrest and growth inhibition. This was supported by the present study, which showed that exposure of BGC-823 cells to (+)-chabranol resulted in an increase in the proportion of cells in the G₀/G₁ phase and a decrease in the proportion of S phase cells, causing cell-cycle arrest in the G₁ phase.

Transmission electron microscopic analysis of the subcellular structure showed that cells exposed to (+)-chabranol were filled with autophagy vacuoles and lipid droplets. This suggested that nutrition of the tumour cells was impaired, and that autophagy provided an alternative source of nutrition to delay tumour degeneration. Autophagy is common feature of cancer cells but the relationship is complex.^29,30 It has been proposed that autophagy is linked to autonomous growth stimuli, insensitivity to antiproliferative signals, disrupted apoptosis and uncontrolled replication.^31,32 Autophagy is also associated with the production of angiogenic factors, metastatic tissue invasion,²⁹ prevention of immune responses³² and enhanced anabolic metabolism.^31,33 Taken together, these factors suggest that the molecular mechanisms of apoptosis and autophagy may proceed in a complex manner. When cell damage exceeds the degree of protection afforded by autophagy, quasiapoptotic factors in the mitochondria may activate the processes involved in cell, death and promote apoptosis.³⁴ These changes might result from mitochondrial membrane damage, whereby increased mitochondrial membrane permeability and reduction in the membrane potential may induce tumour-cell autophagy and apoptosis.³⁵ Further research is required to substantiate this hypothesis.

The present study demonstrated that (+)-chabranol has marked antitumour activity in human gastric cancer BGC-823 cells, and may exert its action by inhibition of cell proliferation and induction of cell apoptosis and autophagy. These findings suggest that (+)-chabranol may be a potential candidate for further study and a drug development target for the treatment of poorly differentiated gastric cancer.

Footnotes

Acknowledgements

This research was supported by grants from: the National Natural Science Fund Project (Nos 30772068 and 81071701); the Key Laboratory of Digestive System Tumours, Gansu Province and the Fundamental Research Fund for Central Universities (lzujbky-2011-t03-15); the Natural Science Foundation of Gansu Province, China (3ZS061-A25-084). The authors wish to thank XL Wang for providing the chabranol and G Chen, J Li and other colleagues for their valuable comments in the development of this manuscript.

The authors had no conflicts of interest to declare in relation to this article.

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