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Cellular Responses to Cancer Chemopreventive Agent D,L-Sulforaphane in Human Prostate Cancer Cells Are Initiated by Mitochondrial Reactive Oxygen Species

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Abstract

Purpose

Present study was undertaken to elucidate the mechanism of cellular responses to D,L-sulforaphane (SFN), a highly promising cancer chemopreventive agent.

Methods

Mitochondrial DNA deficient Rho-0 variants of LNCaP and PC-3 cells were generated by culture in the presence of ethidium bromide. Apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation and activation of caspase-3. Immunoblotting was performed to determine the expression of apoptosis- and cell cycle-regulating proteins. Generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and cell cycle distribution were measured by flow cytometry.

Results

The Rho-0 variants of LNCaP and PC-3 cells were significantly more resistant to SFN-induced ROS generation, apoptotic DNA fragmentation, disruption of MMP, cytosolic release of cytochrome c, and G2/M phase cell cycle arrest compared with corresponding wild-type cells. SFN-induced autophagy, which serves to protect against apoptotic cell death in PC-3 and LNCaP cells, was also partially but markedly suppressed in Rho-0 variants compared with wild-type cells. SFN statistically significantly inhibited activities of mitochondrial respiratory chain enzymes in LNCaP and PC-3 cells.

Conclusion

These results indicate, for the first time, that mitochondria-derived ROS serve to initiate diverse cellular responses to SFN exposure in human prostate cancer cells.

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Correspondence to Shivendra V. Singh.

Additional information

This investigation was supported in part by the USPHS grants CA115498 and CA101753 (to S.V.S.), awarded by the National Cancer Institute, and grant 2718/P01/2007/32 (to A.H-A.), awarded by the Polish Ministry of Science and Higher Education.

Dong Xiao and Anna A. Powolny contributed equally.

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Fig. S1

The SFN-induced recruitment of LC3 to autophagosomes is inhibited by pretreatment with NAC. A, GFP fluorescence in PC-3 cells stably producing GFP-LC3 protein, treated with DMSO or 40 μmol/L SFN for 6 h in the absence (upper pictures) or presence (lower pictures) of 4 mmol/L NAC (2-h pretreatment). In DMSO-treated cells GFP-LC3 is uniformly distributed. SFN treatment induces redistribution of GFP-LC3 to autophagosomes, which is suppressed by NAC. B, The percentage of cells with GFP-LC3 foci treated with DMSO or 40 μmol/L SFN for 6 h in the absence or presence of 4 mmol/L NAC (2-h pretreatment). A total of 200 cells were scored in two separate slides of a given sample. Representative data from a single experiment, which was repeated with similar results, are shown and mean ± SE. Significantly different (P < 0.05) compared with aDMSO-treated control and bSFN only-treated cells by one-way ANOVA followed by Tukey’s test (GIF 46.9 KB).

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Xiao, D., Powolny, A.A., Antosiewicz, J. et al. Cellular Responses to Cancer Chemopreventive Agent D,L-Sulforaphane in Human Prostate Cancer Cells Are Initiated by Mitochondrial Reactive Oxygen Species. Pharm Res 26, 1729–1738 (2009). https://doi.org/10.1007/s11095-009-9883-5

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