Novel camphor-based pyrimidine derivatives induced cancer cell death through a ROS-mediated mitochondrial apoptosis pathway

Abstract

A series of novel camphor-based pyrimidine derivatives (3a-3x) have been synthesized; their structures were determined by using conventional methods and compound 3f was further confirmed through single crystal XRD analysis. The cytotoxic activity of the target compounds against a panel of human normal (GES-1) and cancer cell lines (MDA-MB-231, RPMI-8226, A549) was evaluated by MTS assay. Here we found that compound 3f exhibited the strongest anti-tumor activity, comparable to that of etoposide, and had much lower cytotoxicity to normal GES-1 cells (IC₅₀ > 50 μM) than the reference drug (IC₅₀ = 8.89 μM). Subsequent mechanism studies in MDA-MB-231 cells revealed that compound 3f caused G0/G1 phase arrest and apoptosis in a dose dependent manner. Moreover, the loss of mitochondrial membrane potential and enhancement of cellular ROS levels were also observed upon 3f treatment, which indicated that 3f exerted cytotoxic activity by a ROS-mediated mitochondrial apoptosis pathway. This result was confirmed by a significant increase in the expression of pro-apoptotic proteins Bax, cytochrome C and caspase-3, and downregulation of anti-apoptosis protein Bcl-2. Overall, 3f can be adopted for further investigation in the development of antitumor agents based on natural products.

Scheme 1. Synthesis of compounds 3a–3x.

Fig. 1. X-ray crystal structure of compound 3f.

Fig. 2. The cell viability of synthesized compounds against (a) MDA-MB-231 (b) RPMI-8226 (c) A549 cancer cell line at testing concentrations of 10 μM and 50 μM. The dashed line across the graph represents the threshold percentage for further IC₅₀ determination. Cell viability was measured by MTS assay.

Fig. 3. Influence of compound 3f on cell cycle distribution in MDA-MB-231 cells. (a) Cells were treated with different concentrations (2.5, 5 and 10 μM) of compound 3f for 72 h, which then were stained with propidium iodide (PI), and the DNA content were analyzed by flow cytometry. (b) Percentages of cells in various phases of the cell cycle. These data are from three independent experiments. *p < 0.05; **p < 0.01, verse the negative control.

Fig. 4. Compound 3f induced apoptosis in MDA-MB-231 cells. (a) Flow cytometry analysis of MDA-MB-231 cells were treated with compound 3f (2.5, 5 and 10 μM) for 72 h, respectively, and stained with Annexin V-APC/7-AAD. (b) Quantitative analysis of apoptotic cells in histogram. These data are from three independent experiments.

Fig. 5. Effect of compound 3f on ROS production. (a) Analysis of the ROS levels by flow cytometry after the MDA-MB-231 cells were treated with compound 3f at indicated concentrations or in presence of NAC for 72 h and stained with DCFH-DA. (b) Quantification of the flow cytometric results. These data are from three independent experiments. *p < 0.05 verse the negative control.

Fig. 6. Determination of mitochondrial membrane potential in MDA-MB-231 cells. (a) Cells were treated with compound 3f (2.5, 5 and 10 μM) for 72 h and observed the changes in mitochondrial membrane potential by flow cytometry. (b) Bar graph represents the percentage distribution of cells with red fluorescence or green fluorescence. These data are from three independent experiments.

Fig. 7. Expression changes of apoptosis related proteins. (a) Western blot analysis of Bcl-2, Bax, cytochrome C, caspase-3 in MDA-MB-231 cells treated with compound 3f (2.5, 5 and 10 μM) for 72 h, β-actin was used as the internal reference. (b) Statistical analysis of protein expression levels. These data are from three independent experiments. *p < 0.05 verse the negative control.