Yellow and green pigments from Calophyllum inophyllum L. seed oil induce cell death in colon and lung cancer cells

Abstract

Natural compounds have been candidates for anticancer medicine over the last 20 years. During the process of isolating seed oil from Calophyllum inophyllum L., yellow and green pigments containing multiple compounds with an aromatic structure were identified. High-performance liquid chromatography and nuclear magnetic resonance analysis of these pigments revealed that the compounds present were identical, but the concentration of the compounds was different. Treatment with the pigments was able to induce the death of DLD-1 human colon cancer cells and increase the percentage of the cells in the sub-G₁ and sub-G₂/M phases in a dose-dependent manner. Additionally, the pigments were able to exhibit cytotoxic activity on A549 and H1975 human non-small cell lung cancer (NSCLC) cell lines at 24 h, with half-maximal inhibitory concentrations (IC₅₀) values of 0.1206 and 0.0676%, respectively for green pigments, and 0.0434 and 0.0501%, respectively for yellow pigments. Furthermore, a decrease in IC₅₀ value was associated with an increase in the duration of treatment. However, a sharp decrease in IC₅₀ value of the yellow pigment was observed for H1975 cells at 48 h and for A549 cells at 72 h compared with no change in IC₅₀ value for the green pigment with time, suggesting that the pigments function and induce cell death differently in the two cell lines. An investigation was performed into the synergistic effect of the green pigment and gefitinib (Iressa^®, ZD1839), which is a selective epidermal growth factor receptor-tyrosine kinase inhibitor to block growth factor-mediated cell proliferation. The combination of the green pigment and gefitinib resulted in an enhancement of the decrease in viability of A549 and H1975 cells compared with treatment with gefitinib alone, which suggested that treatment with the green pigments was able to enhance the sensitivity of NSCLC cells to gefitinib. In conclusion, these pigments may be considered for development as anti-colon cancer agents.

Keywords: apoptosis; colon cancer; gefitinib; high-performance liquid chromatography; non-small cell lung cancer; pigments.

Figure 1.

Nuclear magnetic resonance analysis of (A) yellow and (B) green pigments from the seed oil obtained from Calophyllum inophyllum L. ppm, parts per million.

Figure 2.

High-performance liquid chromatography analysis of (A) yellow and (B) green pigments from the seed oil obtained from Calophyllum inophyllum L. AU, absorption units.

Figure 3.

Cell viability analysis in green or yellow pigment-treated DLD-1 cells. DLD-1 cells were plated in 60 mm culture dishes at 80% confluence and treated with the indicated concentrations of pigment for 24 h. Following treatment, the MTT reagent (0.5 mg/ml) was added to the cells for 2 h at 37°C, and the cells were lysed with dimethyl sulfoxide. Absorbance was evaluated at 595 nm. IC₅₀, half-maximal inhibitory concentration.

Figure 4.

(A) Apoptotic analysis of green or yellow pigment-treated DLD-1 cells. ***P<0.001 vs. untreated. (B) Cell cycle analysis of green or yellow pigment-treated DLD-1 cells. DLD-1 cells were plated in 60 mm culture dishes at 80% confluence and treated with the indicated concentrations of pigment for 24 h. Following treatment, the cells were harvested and fixed in phosphate-buffered saline-methanol (v/v, 1:2) solution. The cells were stained with propidium iodide followed by flow cytometric analysis. *P<0.05, **P<0.01 and ***P<0.001 vs. untreated.

Figure 5.

Green pigment induces cytotoxicity in human lung cancer cells. Various concentrations (0.05, 0.10, 0.25, 0.50 and 1.00%) of the pigment were added to A549 or H1975 cells for 24–72 h. Cell viability was determined by MTS assay. NSCLC, non-small cell lung cancer.

Figure 6.

Yellow pigment induces cytotoxicity in human lung cancer cells. Various concentrations (0.05, 0.10, 0.25, 0.50 and 1.00%) of the pigments were added to A549 or H1975 cells for 24–72 h. Cell viability was determined by MTS assay. NSCLC, non-small cell lung cancer.