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, 2013, 857257

Induction of Apoptosis and Cell Cycle Blockade by Helichrysetin in a549 Human Lung Adenocarcinoma Cells

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Induction of Apoptosis and Cell Cycle Blockade by Helichrysetin in a549 Human Lung Adenocarcinoma Cells

Yen Fong Ho et al. Evid Based Complement Alternat Med.

Abstract

Researchers are looking into the potential development of natural compounds for anticancer therapy. Previous studies have postulated the cytotoxic effect of helichrysetin towards different cancer cell lines. In this study, we investigated the cytotoxic effect of helichrysetin, a naturally occurring chalcone on four selected cancer cell lines, A549, MCF-7, Ca Ski, and HT-29, and further elucidated its biochemical and molecular mechanisms in human lung adenocarcinoma, A549. Helichrysetin showed the highest cytotoxic activity against Ca Ski followed by A549. Changes in the nuclear morphology of A549 cells such as chromatin condensation and nuclear fragmentation were observed in cells treated with helichrysetin. Further evidence of apoptosis includes the externalization of phosphatidylserine and the collapse of mitochondrial membrane potential which are both early signs of apoptosis. These signs of apoptosis are related to cell cycle blockade at the S checkpoint which suggests that the alteration of the cell cycle contributes to the induction of apoptosis in A549. These results suggest that helichrysetin has great potentials for development as an anticancer agent.

Figures

Figure 1
Figure 1
(a) Structure of helichrysetin. (b) HT-29, A549, MCF-7, and Ca Ski cells were treated with different concentrations of helichrysetin for up to 72 hours, and the percentage of inhibition was evaluated using MTT assay. The results are shown as mean ± S.E. and P < 0.05 was regarded as statistically significant compared to the untreated control. The percentage of inhibition in untreated control was normalized to 0%. (c) DAPI staining on untreated A549 cells (left) and helichrysetin-treated A549 cells (right) at 40x magnification. Nuclear fragmentation is indicated by bright blue cells (arrow).
Figure 2
Figure 2
(a) Untreated control was compared with cells treated at different doses and time. Cells' morphological changes were observed under phase-contrast microscopy at 40x magnification. Cells shrinkage and rounding were observed clearly. (b) JC-1 dye was used for the analysis of cell mitochondrial membrane potential for flow cytometry as described in Section 2. Cells treated with helichrysetin were found to have lost mitochondrial membrane potential as measured by loss in fluorescence.
Figure 3
Figure 3
(a) Helichrysetin increases apoptotic cells significantly at 15 μg/mL and 20 μg/mL. Results are presented as mean ± S.E., and values of P < 0.05 were regarded as statistically significant compared to the untreated control. After treatment for 24 h, 48 h, and 72 h, an increase in the percentage of apoptotic cells was observed. ((b)(i)) A549 cells were treated with helichrysetin at 5 μg/mL, 15 μg/mL, and 20 μg/mL. ((b)(ii)) A549 cells treated for 24 h, 48 h, and 72 h (bottom). Lower left quadrants show viable cells, lower right quadrants show early apoptotic cells, and upper right quadrants show late apoptotic/necrotic cells.
Figure 4
Figure 4
(a) TUNEL assay detects DNA fragmentation in cells. Results showed that the percentage of TUNEL-positive cells increased significantly in a dose- and time-dependent manner, presented as mean ± S.E., and values of P < 0.05 were regarded as statistically significant compared to the untreated control. (b) A549 cells were treated with helichrysetin at 5 μg/mL, 15 μg/mL, and 20 μg/mL (top) and 24 h, 48 h, and 72 h (bottom). Density plots show the increase of Alexa Fluor fluorescence intensity as the dose and time increases.
Figure 5
Figure 5
(a) Cells were treated with helichrysetin at different concentrations.After 72 hours of treatment, results showed the cell cycle distributions which have been summarized and presented as mean ± S.E., and values of P < 0.05 were regarded as statistically significant compared to the untreated control. *P < 0.05. (b) Cell cycle distribution of untreated cells. (c) Cell cycle distribution at 5 μg/mL. (d) Cell cycle distribution at 15 μg/mL. (e) Cell cycle distribution at 20 μg/mL.

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