Gamma-tocotrienol and hydroxy-chavicol synergistically inhibits growth and induces apoptosis of human glioma cells

Abstract

Background: Gamma-tocotrienol (GTT), an isomer of vitamin E and hydroxy-chavicol (HC), a major bioactive compound in Piper betle, has been reported to possess anti-carcinogenic properties by modulating different cellular signaling events. One possible strategy to overcome multi-drug resistance and high toxic doses of treatment is by applying combinational therapy especially using natural bioactives in cancer treatment.

Methods: In this study, we investigated the interaction of GTT and HC and its mode of cell death on glioma cell lines. GTT or HC alone and in combination were tested for cytotoxicity on glioma cell lines 1321N1 (Grade II), SW1783 (Grade III) and LN18 (Grade IV) by [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)- 2H- tetrazolium, inner salt] MTS assay. The interactions of each combination were evaluated by using the combination index (CI) obtained from an isobologram.

Results: Individually, GTT or HC displayed mild growth inhibitory effects against glioma cancer cell lines at concentration values ranging from 42-100 μg/ml and 75-119 μg/ml respectively. However, the combination of sub-lethal doses of GTT + HC dramatically enhanced the inhibition of glioma cancer cell proliferation and exhibited a strong synergistic effect on 1321N1 with CI of 0.55, and CI = 0.54 for SW1783. While in LN18 cells, moderate synergistic interaction of GTT + HC was observed with CI value of 0.73. Exposure of grade II, III and IV cells to combined treatments for 24 hours led to increased apoptosis as determined by annexin-V FITC/PI staining and caspase-3 apoptosis assay, showing caspase-3 activation of 27%, 7.1% and 79% respectively.

Conclusion: In conclusion, combined treatments with sub-effective doses of GTT and HC resulted in synergistic inhibition of cell proliferation through the induction of apoptosis of human glioma cells in vitro.

Figure 1

Treatment of (a) gamma-tocotrienol (GTT), (b) hydroxyl-chavicol (HC) and (c) combination of GTT with HC; on 1321N1, SW1783 and LN18 for 24 h. The cell survival test was determined by MTS assay. Data is presented as means ± SD, n = 9. *P < 0.05 compare to untreated 1321N1; # P < 0.05 compare to untreated SW1783; $P < 0.05 compare to untreated LN18.

Figure 2

Morphological changes of 1321N1, SW1783 and LN18 cells after treatment. a. Morphological changes of 1321N1 cells after treatment with (i) vehicle untreated cells (ii) GTT 40 μg/ml, (iii) HC 12 μg/ml, (iv) 40 μg/ml of GTT + 12 μg/ml of HC, (v) scale 100 μm; 40 μg/ml of GTT + 12 μg/ml of HC; for 24 h. Arrow shows; x. chromatin margination (cresents), y. the collapse of nuclease and vacuolized cytoplasm, and z. cell shrinkage. b. Morphological changes of SW1783 cells after treatment with (i) vehicle untreated cells (ii) GTT 40 μg/ml, (iii) HC 2 μg/ml, (iv) 40 μg/ml of GTT + 2 μg/ml of HC, (v) scale 100 μm; 40 μg/ml of GTT + 2 μg/ml of HC; for 24 h. Arrow represents; x. collapse of nuclease, y. volume loss and chromatin clumping, and z. cell shrinkage. c. Morphological changes of LN18 cells after treatment with (i) vehicle untreated cells (ii) GTT 20 μg/ml, (iii) HC 29 μg/ml, (iv) 20 μg/ml of GTT + 29 μg/ml of HC, (v) scale 100 μm; 20 μg/ml of GTT + 29 μg/ml of HC; for 24 h. Arrow represents x. volume loss and chromatin clumping, y. collapse of nuclease, and z. cell shrinkage.

Figure 3

Isobologram analysis of GTT and HC antiproliferative effects on glioma cells. Individual IC50 doses for GTT and HC were calculated and then plotted on the x and y axes. The line connecting these points represents the drug doses of each compound that would induce the same growth inhibition when used in combination if the interaction between these compounds were additive. The data point on the isobologram represents the actual doses of combined GTT and HC treatment that results in 50% growth inhibition. Since the data point is positioned well below the line, a synergistic anti-proliferative effect is indicated.

Figure 4

Detection of apoptosis using flow cytometry after caspase-3 antibody staining. 1321N1, SW1783 and LN18 cells were treated with combined treatments at IC50 concentrations for 24 h. (A) An example of active caspase-3 detection diagram from 1321N1 cells: viable cells are in the left quadrant, while the presence of active caspase-3 is shown in right quadrant (P2). (B) Combined compounds caused greater inhibition of growth of 1321N1, SW1783 and LN18 cells than either agent alone as evidence by the presence of active caspase-3. Each value represents mean ± SD of three independent experiments. *P < 0.05 compare to control. # P < 0.05 compare to GTT. $P < 0.05 compare to HC.

Figure 5

Combined treatments potentiate apoptosis mediated cell death in 1321N1, SW1783 and LN18 cells. (A) Detection of apoptosis using flow cytometry after annexin V-FITC/propidium iodide (PI) staining. 1321N1, SW1783 and LN18 cells were treated with combined treatments at IC50 concentrations for 24 h. Viable cells are in the lower left quadrant (Q3), early apoptotic cells are in the lower right quadrant (Q4), late apoptotic cells are in the upper right quadrant and non-viable necrotic cells are in the upper left quadrant (Q1). (B) Bar graph representing mean values from three independent experiments for i.1321N1, ii. SW1783 and iii. LN18. *P < 0.05 compare to control early or late apoptosis respectively. # P < 0.05 compare to GTT early or late apoptosis respectively. $P < 0.05 compare to HC early or late apoptosis respectively.

Figure 6

Bar graph of normal foreskin fibroblast and normal liver WRL68 cells treated with combined GTT + HC after 24 hours. No sign of toxicity were observed. Data represent the mean ± SD (n = 3).