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Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways - PubMed

Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways

Chuan-bin Yang et al. Acta Pharmacol Sin. 2014 Jan.

Abstract

Aim: The purpose of the present study was to investigate the anticancer activity of bornyl caffeate in the human breast cancer cell line MCF-7.

Methods: The cell viability was determined using the MTT assay, and apoptosis was initially defined by monitoring the morphology of the cell nuclei and staining an early apoptotic biomarker with Annexin V-FITC. The mitochondrial membrane potential was visualized by JC-1 under fluorescence microscopy, whereas intracellular reactive oxygen species (ROS) were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis.

Results: Bornyl caffeate induced apoptosis in MCF-7 cells in a dose- and time-dependent manner. Consistently, bornyl caffeate increased Bax and decreased Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and the activation of the mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). Antioxidants attenuated the activation of MAP kinase p38 but barely affected the activation of JNK. Importantly, the cytotoxicity of bornyl caffeate was partially attenuated by scavenging ROS and inhibited by MAP kinases and caspases.

Conclusion: The present study demonstrated that bornyl caffeate induced apoptosis in the cancer cell line MCF-7 via activating the ROS- and JNK-mediated pathways. Thus, bornyl caffeate may be a potential anticancer lead compound.

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Figures

Figure 1
Figure 1

Scheme illustrating the chemical synthesis of bornyl caffeate. Bornyl caffeate was synthesized via acid-catalyzed esterification of caffeic acid with borneol. The structures were generated using the chemistry software ACD/ChemSketch (

http://www.acdlabs.com

).

Figure 2
Figure 2

Effect of bornyl caffeate on the cell viability of human breast cancer MCF-7 cells. (A) Dose-dependence of cytotoxicity. The cells were treated for 24 h with bornyl caffeate at the indicated doses. (B) Time-dependence of cytotoxicity. The cells were treated with 50 μmol/L of bornyl caffeate at each time point, whereas the control cells were treated with DMSO. At the end of drug treatment, the cell viability was determined using the MTT assay. The results are expressed as a percentage of the corresponding control. (C) Dose-dependence of LDH release. The cells were treated for 24 h with bornyl caffeate at the indicated doses. (D) Time-dependence of LDH release. The cells were treated with 50 μmol/L of bornyl caffeate at each time point, whereas the control cells were treated with DMSO. At the end of drug treatment, the cell viability was determined using the MTT assay, and the results are expressed as a percentage of the corresponding control. Mean±SD. n=3. bP<0.05, cP<0.01 vs control.

Figure 3
Figure 3

Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells. (A) Effect of bornyl caffeate on the morphology of cell nuclei. MCF-7 cells were treated with 0, 10, 25, or 50 μmol/L of bornyl caffeate for 24 h and subsequently stained with Hoechst 33258, and the images were captured by fluorescence microscopy. The arrows point to the condensed and/or fragmented cell nuclei. (B) Flow cytometric analysis of the cell-surface phosphoserine. MCF-7 cells were sequentially treated with 0, 10, 25, or 50 μmol/L bornyl caffeate for 24 h, co-stained with Annexin V-FITC and PI and analyzed by flow cytometry (FACSCalibur, BD Biosciences, USA). (C) Effect of bornyl caffeate on the mitochondrial membrane potential. Cells were treated with 0, 10, 25, or 50 μmol/L of bornyl caffeate for 24 h and subsequently stained with a fluorescent dye DJ-1. The cells were visualized for their green and red emission components by using optical filters designed for fluorescein and tetramethylrhodamine fluorescence microscopy. The arrows point to the cells undergoing depolarization of the mitochondrial membrane. (D) Effect of bornyl caffeate on the formation of ROS. Cells were treated with 0, 10, 25, and 50 μmol/L of bornyl caffeate for 24 h. The ROS levels were determined by assaying the fluorescent product 2′,7′-dichlorofluorescein (DCF) from 2′,7′-dichlorofluorescin diacetate (DCFH-DA) in viable cells on a flow cytometer (FACSCalibur, BD Biosciences, USA).

Figure 4
Figure 4

Effect of bornyl caffeate on the expression and bioprocessing of apoptosis-related biomarkers. (A) Western blot analysis of Bax, Bcl-2, and Bcl-xL. MCF-7 cells were treated with bornyl caffeate at concentrations of 0, 10, 25, or 50 μmol/L for 24 h. Bax, Bcl-xL, and Bcl-2 were detected by specific antibodies, whereas GAPDH was detected as the internal control. (B) Data quantification of Panel A. The Western blots were quantified and expressed as the fold change of signal intensity compared to the untreated sample after normalization to the corresponding GAPDH signals. (C) Western blot analysis of caspase-3 and PARP cleavage. At the end of treatment, the cellular proteins were analyzed for caspase-3 and PARP cleavage by specific antibodies in the same way as described in Panel A. (D) Data quantification of Panel C. The data were analyzed in the same way as described in Panel B. Mean±SD. n=3. bP<0.05, cP<0.01 vs control.

Figure 5
Figure 5

Bornyl caffeate differentially activated MAP kinases in MCF-7 cells. (A) Effect of bornyl caffeate on MAP kinases. MCF-7 cells were treated with 50 μmol/L bornyl caffeate for the indicated times. Total cell lysates were extracted, resolved by 10% SDS-PAGE and transferred onto PVDF membrane. The phosphorylated and total proteins of Akt, ERKs, p38 MAPK, and JNKs were immunodetected. (B) Quantification of Panel A. The Western blots were quantified and expressed as the fold change of the signal intensity value compared to the untreated sample after normalization to the corresponding GAPDH signals. (C) Effects of antioxidants on the bornyl caffeate-induced activation of MAP kinases. MCF-7 cells were pretreated with 1 mmol/L of different antioxidants, except for vitamin C (100 μmol/L), for 1 h followed by treatment with 50 μmol/L bornyl caffeate for 24 h. The intracellular proteins were extracted and analyzed by Western blot using specific antibodies. Mean±SD. n=3. bP<0.05, cP<0.01 vs control.

Figure 6
Figure 6

Investigation of potential mechanisms underlying the cytotoxicity of bornyl caffeate. (A) Effect of protein kinase inhibitors on the cytotoxicity of bornyl caffeate. MCF-7 cells were pretreated with different protein kinase inhibitors (PI3 kinase inhibitor LY294002, ERK inhibitor PD98059, p38 MAP kinase inhibitor SP600125 or JNK inhibitor SB203580) for 1 h followed by treatment with 50 μmol/L bornyl caffeate for 24 h. Cell viability was determined using the standard MTT assay. (B) Effect of different antioxidants on the cytotoxicity of bornyl caffeate. MCF-7 cells were pretreated with 1 mmol/L of different antioxidants, except for vitamin C (100 μmol/L), for 1 h followed by treatment with 50 μmol/L bornyl caffeate for 24 h. The cell viability was determined using the standard MTT assay. (C) Effect of the pan-caspase inhibitor z-VAD-fmk on the cytotoxicity of bornyl caffeate. MCF-7 cells were pretreated with 50 μmol/L z-VAD-fmk for 1 h followed by treatment with 50 μmol/L bornyl caffeate for 24 h. The cell viability was determined using the standard MTT assay (bP<0.05, cP<0.01 vs control).

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