The apoptotic effect of 1's-1'-acetoxychavicol acetate from Alpinia conchigera on human cancer cells

Abstract

1'-(S)-1'-Acetoxychavicol acetate (ACA) isolated from the Malaysian ethno-medicinal plant Alpinia conchigera Griff. was investigated for its potential as an anticancer drug. In this communication, we describe the cytotoxic and apoptotic properties of ACA on five human tumour cell lines. Data from MTT cell viability assays indicated that ACA induced both time- and dose-dependent cytotoxicity on all tumour cell lines tested and had no adverse cytotoxic effects on normal cells. Total mortality of the entire tumour cell population was achieved within 30 hrs when treated with ACA at 40.0 µM concentration. Flow cytometric analysis for annexin-V and PI dual staining demonstrated that cell death occurred via apoptosis, followed by secondary necrosis. The apoptotic effects of ACA were confirmed via the DNA fragmentation assay, in which consistent laddering of genomic DNA was observed for all tumour cell lines after a 24 hrs post-treatment period at the IC(50) concentration of ACA. A cell cycle analysis using PI staining also demonstrated that ACA induced cell cycle arrest at the G(0)/G(1) phase, corresponding to oral tumour cell lines. In conclusion, ACA exhibits enormous potential for future development as a chemotherapeutic drug against various malignancies.

Figure 1

Chemical structure of 1’S-1’-acetoxychavicol acetate.

Figure 2

Chromatogram of 1’S-1’-acetoxychavicol acetate at 254 nm.

Figure 3

The cytotoxic effects of ACA on tumour cell lines assessed using the MTT cell viability assay. (A) Comparison of total viable cell count between various tumour cell lines after treatment with ACA at different concentrations (0–80.0 μM) at 12 hrs post-treatment time. (B) Comparison of total viable cell count between various tumor cell lines after treatment with 40.0 μM ACA for 30 hrs. All experiments were plotted as mean values (n = 3).

Figure 4

Cell cycle distribution of cancer cell lines using flow cytometry after staining with PI. (A) HSC-2 cells treated with ACA; (B) HSC-4 cells treated with ACA. M1: Sub-G₁ phase; M2:G₀/G₁ phase; M3:S phase; M4:G₂/M phase. All experiments are a representative of 10,000 cells and the percentage of cells in all cell cycle phases are indicated.

Figure 5

Detection of apoptosis using flow cytometry after annexin V-FITC/ propidium iodide (PI) staining indicating that ACA potentiates apoptosis-mediated cell death after 12 hrs and 24 hrs of exposure. (A) HSC-2 cells treated with ACA; (B) HSC-4 cells treated with ACA. Viable cells are in the lower left quadrant, early apoptotic cells are in the lower right quadrant, late apoptotic cells are in the upper right quadrant and non-viable necrotic cells are in the upper left quadrant. Dot plots are a representative of 10,000 cells from a single replicate.

Figure 6

Confirmation of apoptosis using the DNA fragmentation assay. Cells were both untreated and treated with ACA for 12 hrs and 24 hrs to observe its effects on DNA laddering (M: 100 bp DNA Size Marker).