Oleandrin induces DNA damage responses in cancer cells by suppressing the expression of Rad51

Abstract

Oleandrin is a monomeric compound extracted from leaves and seeds of Nerium oleander. It had been reported that oleandrin could effectively inhibit the growth of human cancer cells. However, the specific mechanisms of the oleandrin-induced anti-tumor effects remain largely unclear. Genomic instability is one of the main features of cancer cells, it can be the combined effect of DNA damage and tumour-specific DNA repair defects. DNA damage plays important roles during tumorigenesis. In fact, most of the current chemotherapy agents were designed to kill cancer cells by inducing DNA damage. In this study, we found that oleandrin was effective to induce apoptosis in cancer cells, and cause rapid DNA damage response, represented by nuclear RPA (Replication Protein A, a single strand DNA binding protein) and γH2AX(a marker for DNA double strand breaks) foci formation. Interestingly, expression of RAD51, a key protein involved in homologous recombination (HR), was suppressed while XRCC1 was up-regulated in oleandrin treated cancer cells. These results suggested that XRCC1 may play a predominant role in repairing oleandrin-induced DNA damage. Collectively, oleandrin may be a potential anti-tumor agent by suppressing the expression of Rad51.

Keywords: DNA damage response; DNA replication; apoptosis; oleandrin; single strand break repair.

Figure 1. Oleandrin induced apoptosis in A549 cells

A. Representative FACS profiles of A549 cells treated with different concentrations of oleandrin for 24 hours. B. Quantification of Annexin V positive and PI positive A549 cells following treatment of oleandrin at indicated concentrations were shown. C. Representative FACS profiles of A549 cells treated with oleandrin (0.02 ug/ml) for different hours (6h, 12h, 24h). D. Quantification of Annexin V positive and PI positive A549 cells following treatment of oleandrin for indicated hours were shown. All data represent mean ± SEM (n=3); n.s. not significant, ***P<0.001, compared with the controls.

Figure 2. Oleandrin effectively induced cell death in A549 and H1299 cells, but not in HBE and ATDC5 cells

A. Quantification of cell death in A549 cells treated with oleandrin (0.02ug/ml) for 24 hours, detected by FACS. B. Quantification of cell death in H1299 cells treated with oleandrin (0.02ug/ml) for 24 hours, detected by FACS. C. Quantification of cell death in HBE cells treated with oleandrin (0.02ug/ml) for 24 hours, detected by FACS. D, E. Quantification of cell viability in A549 cells and ATDC5 cells treated with oleandrin (0.02ug/ml) for 24 hours, detected by CCK-8 assay. All data represent mean±SEM(n=3); n.s. not siginificant; **P<0.01; ***P<0.001, compared with controls.

Figure 3. Oleandrin induced RPA and gH2AX foci in lung cancer cells

A. Immunofluorescence staining of RPA in A549 cells treated with oleandrin (0.02ug/ml) for 2hours, 6hours, 12hours. B. Quantification of (A). C. Immunofluorescence staining of RPA in H1299 cells treated with oleandrin (0.02ug/ml) for 2hours, 6hours, 12hours. D. Quantification of (C). E. Immunofluorescence staining of γH2AX in A549 cells treated with oleandrin (0.02ug/ml) for 24hours. F. A549 cells treated with oleandrin (0.02ug/ml) for 24hours, γH2AX was analyzed by western blot. All data represent mean±SEM (n=3). ***P<0.001, compared with controls.

Figure 4. Expression of DNA damage repair proteins in cancer cell lines following treatment with oleandrin

A. Western blot analysis of RAD51 and Actin in A549 cells. Quantification of RAD51 expression level was performed by densitometric analysis. B. Western blot analysis of RAD51 and Actin in H1299 cells. Quantification of RAD51 expression level was performed by densitometric analysis. C. Western blot analysis of XRCC1 in A549 cells. Quantification of XRCC1 expression level was performed by densitometric analysis. D. Cell cycle of A549 cells treated with oleandrin (0.02ug/ml) for 24 hours. Percentage of cells stalled in G2/M phase were quantified. All data represent mean±SEM (n=3). n.s. not significant; **P<0.01; ***P<0.001, compared with controls.

Figure 5. Loss of XRCC1 sensitized A549 cells to oleandrin-induced cell death

A. Western blot analysis of XRCC1 and Actin in A549 cells following indicated siRNA transfection. B. A549 cells were incubated with oleandrin (0.02ug/ml) for 12 hours, 24 hours after the transfection of XRCC1 siRNA. Cell death was detected with FACS analysis. C, D, E. Quantification of (B) for indicated values. All data represent mean±SEM (n=3). ***P<0.001.