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. 2018 Apr 27;19(5):1309.
doi: 10.3390/ijms19051309.

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells

Seon Min Woo  1 , Seung Un Seo  2 , Kyoung-Jin Min  3 , Seung-Soon Im  4 , Ju-Ock Nam  5 , Jong-Soo Chang  6 , Shin Kim  7 , Jong-Wook Park  8 , Taeg Kyu Kwon  9
Affiliations

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells

Seon Min Woo et al. Int J Mol Sci. .

Abstract

Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants (N-acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

Keywords: corosolic acid; lipid ROS; non-apoptotic cell death; α-tocopherol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Corosolic acid induces non-apoptotic cell death through caspase-independent manner. (A,B) Caki cells were treated with 2.5, 5, or 10 µM corosolic acid for 24 h. 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used to detect the cell viability (A); Lactate dehydrogenase (LDH) release assay was used to detect the cell cytotoxicity (B); (C) Caki cells were treated with 10 µM corosolic acid for 24 h. We detected the cell morphology using interference light microscopy; (D) Caki cells were treated with 10 µM corosolic acid or 10 ng/mL TNF-α plus 5 µg/mL cycloheximide (CHX) for 24 h in the presence or absence of 20 µM z-VAD-fmk (z-VAD). XTT assay was used to detect the cell viability; (E–G) Caki cells were treated with 2.5, 5, or 10 µM corosolic acid for 24 h (p.c: positive control; 10 ng/mL TNF-α plus 5 µg/mL CHX for 24 h). Caspase activities were detected using a kit, as described in material and methods (E); Western blotting was used to detect the protein levels of PARP and actin (F); Flow cytometry was used to detect the Annexin V/7-AAD staining (G); (H) Caki cells were treated with 10 µM corosolic acid for 24 h. After treatment with corosolic acid, cells were stained with propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI), and fluorescence microscope (left panel) or flow cytometry (right panel) was used to detect PI uptake. The values in the graphs (A,B,D,E,H) represent the mean ± SD of three independent samples. * p < 0.01 compared to the control.
Figure 2
Figure 2
Corosolic acid-induced cell death is independent of necroptosis. (A–C) Caki cells were treated with 10 µM corosolic acid or 50 µM artesunate (positive control) in the presence or absence of 60 µM necrostatin-1. We detected the cell morphology using interference light microscopy (A); XTT assay was used to detect the cell viability (B); LDH release assay was used to detect the cell cytotoxicity (C); (D,E) Caki cells were transiently transfected with siRNA against control, RIP1, and AIF. After 24 h, cells were treated with 10 µM corosolic acid for 24 h. LDH release was used to detect the cell cytotoxicity, and western blotting was used to detect the protein levels of RIP1, AIF, and/or actin. The values in the graphs (B,C,D,E) represent the mean ± SD of three independent samples.
Figure 3
Figure 3
The generation of ROS is not involved in the induction of corosolic acid-induced non-apoptotic cell death. (A) Caki cells were treated with 10 µM corosolic acid for 9 h. After treatment with corosolic acid, cells were loaded with H2DCF-DA fluorescent dye, and fluorescence microscope (upper panel) or flow cytometry (lower panel) was used to detect intracellular ROS levels; (B–E) Caki cells were pre-treated with 5 mM NAC and 200 µM trolox for 30 min and were then treated with 10 µM corosolic acid for 9 h (B) and 24 h (C–E); Cells were loaded with H2DCF-DA fluorescent dye, and flow cytometry was used to detect intracellular ROS levels (B); We detected the cell morphology using interference light microscopy (C); XTT assay was used to detect the cell viability (D), and LDH release assay was used to detect the cell cytotoxicity (E); The values in the graphs (B,D,E) represent the mean ± SD of three independent samples. * p < 0.01 compared to corosolic acid-treated cells.
Figure 4
Figure 4
Corosolic acid-induced cell death is not associated with induction of ferroptosis. (A) Caki cells were treated with 10 µM corosolic acid for indicated time periods (left panel) or 9 h (right panel) (p.c: positive control; 2 µM RAS-selective lethal 3 (RSL3) for 9 h). After treatment with corosolic acid, cells were loaded with BODIPY-C11 fluorescent dye, and flow cytometry was used to detect lipid peroxidation; (B–D) Caki cells were pre-treated with 200 µM DFO and 1 µM ferrostatin-1 for 30 min and were then treated with 10 µM corosolic acid or 2 µM RSL3 for 24 h. We detected the cell morphology using interference light microscopy (B); XTT assay was used to detect the cell viability (C), and LDH release assay was used to detect the cell cytotoxicity (D); The values in the graphs (A,C,D) represent the mean ± SD of three independent samples. * p < 0.01 compared to the control.
Figure 5
Figure 5
α-tocopherol inhibits corosolic acid-induced lipid peroxidation and cell death. (A,B) Caki cells were pre-treated with the indicated concentrations of α-tocopherol for 30 min and were then treated with 10 µM corosolic acid for 24 h. We detected the cell morphology using interference light microscopy (A); XTT assay and LDH release assay were used to detect the cell viability and cell cytotoxicity, respectively (B); (C) Caki cells were pre-treated with 200 µM DFO, 1 µM ferrostatin-1, and 100 µM α-tocopherol for 30 min and were then treated with 10 µM corosolic acid or 2 µM RSL3 for 9 h. After treatment with corosolic acid, cells were loaded with BODIPY-C11 fluorescent dye, and flow cytometry was used to detect lipid peroxidation; (D) Caki cells were pre-treated with 100 µM α-tocopherol for 30 min and were then treated with 10 µM corosolic acid for 9 h. After treatment with corosolic acid, cells were loaded with H2DCF-DA fluorescent dye, and flow cytometry was used to detect intracellular ROS levels; (E) Caki cells were pre-treated with 5 mM NAC and 200 µM trolox for 30 min and were then treated with 10 µM corosolic acid or 2 µM RSL3 for 9 h. After treatment, cells were loaded with BODIPY-C11 fluorescent dye, and flow cytometry was used to detect lipid peroxidation. The values in the graphs (B,C,D,E) represent the mean ± SD of three independent samples. * p < 0.01 compared to corosolic acid-treated cells.
Figure 6
Figure 6
The effects of the corosolic acid-induced cell death in other carcinoma and normal cells. (A) Cancer cells were pre-treated with 20 µM z-VAD, 60 µM necrostatin-1, 1 µM ferrostatin-1, and 100 µM α-tocopherol for 30 min and were then treated with 10 µM corosolic acid for 24 h. XTT assay and LDH release assay were used to detect the cell viability (upper panel) and the cell cytotoxicity (lower panel); (B) Caki and mesangial cell (MC) cells were treated with 10 µM corosolic acid for 24 h. XTT assay and LDH release assay were used to detect the cell viability and the cell cytotoxicity; The values in the graphs (A,B) represent the mean ± SD of three independent samples. * p < 0.01 compared to corosolic acid-treated cells.
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References

    1. Devasagayam T.P., Tilak J.C., Boloor K.K., Sane K.S., Ghaskadbi S.S., Lele R.D. Free radicals and antioxidants in human health: Current status and future prospects. J. Assoc. Physicians India. 2004;52:794–804. - PubMed
    1. Boonstra J., Post J.A. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene. 2004;337:1–13. doi: 10.1016/j.gene.2004.04.032. - DOI - PubMed
    1. Toyokuni S., Okamoto K., Yodoi J., Hiai H. Persistent oxidative stress in cancer. FEBS Lett. 1995;358:1–3. doi: 10.1016/0014-5793(94)01368-B. - DOI - PubMed
    1. Bielski B.H., Arudi R.L., Sutherland M.W. A study of the reactivity of HO2/O2- with unsaturated fatty acids. J. Biol. Chem. 1983;258:4759–4761. - PubMed
    1. Gaschler M.M., Stockwell B.R. Lipid peroxidation in cell death. Biochem. Biophys. Res. Commun. 2017;482:419–425. doi: 10.1016/j.bbrc.2016.10.086. - DOI - PMC - PubMed

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