The p53-dependent apoptotic pathway of breast cancer cells (BC-M1) induced by the bis-type bioreductive compound aziridinylnaphthoquinone.
BACKGROUND
Several aziridinylbenzoquinone drugs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill cells preferentially within the hypoxia tumor microenvironment. The bioreductive compound of bis-type naphthoquinone synthesized in our laboratory, 2-aziridin-1-yl-3-[(2-{2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy}ethyl)thio]naphthoquinone (AZ-1), had the most potent death effect on the breast cancer cells BC-M1 in our previous screening. In the present study, we determined that the mechanism of the death effect of BC-M1 cells induced by AZ-1 was mediated by the apoptosis pathway.


METHODS
We evaluated the cytotoxicity of AZ-1 and the anti-breast cancer drugs tamoxifen and paclitaxel to BC-M1 cells and MCF-7 cells by the MTT assay and measured the apoptosis phenomena by Hoechst 33258 staining for apoptotic bodies. We also quantified the sub-G1 peak area and the ratio of the CH2/CH3 peak area of the cell membrane in BC-M1 cells by flow cytometry and 1H-NMR spectra, respectively. The apoptosis-related protein expressions, including p53, p21, the RNA-relating protein T-cell restricted intracellular antigen-related protein, cyclin-dependent kinase 2 (cell cycle regulating kinase) and pro-caspase 3, were detected by western blot, and the caspase-3 enzyme activity was also quantified by an assay kit.


RESULTS
AZ-1 induced two of the breast cancer cell lines, with IC50 = 0.51 microM in BC-M1 cells and with IC50= 0.57 microM in MCF-7 cells, and showed less cytotoxicity to normal fibroblast cells (skin fibroblasts) with IC50= 5.6 microM. There was a 10-fold difference between two breast cancer cell lines and normal fibroblasts. Of the two anti-breast cancer drugs, tamoxifen showed IC50= 0.12 microM to BC-M1 cells and paclitaxel had much less sensitivity than AZ-1. The expression of p53 protein increased from 0.5 to 1.0 microM AZ-1 and decreased at 2.0 microM AZ-1. The p21 protein increased from 0.5 microM AZ-1, with the highest at 2 microM AZ-1. Regarding the AZ-1 compound-induced BC-M1 cells mediating the apoptosis pathway, the apoptotic body formation, the sub-G1 peak area, the ratio of CH2/CH3 of phospholipids in the cell membrane and the enzyme activity of caspase-3 were all in direct proportion with the dose-dependent increase of the concentration of AZ-1. The death effect-related proteins, including T-cell restricted intracellular antigen-related protein, cyclin-dependent kinase 2, and pro-caspase-3, all dose-dependently decreased with AZ-1 concentration.


CONCLUSIONS
The AZ-1-induced cell death of BC-M1 cells mediating the apoptosis pathway might be associated with p53 protein expression, and AZ-1 could have the chance to be a candidate drug for anti-breast cancer following more experimental evidence, such as animal models.
Yang
Yu-Ping
YP
Department of Medicine, College of Medicine, Fu-Jen Catholic University, Taipei Hsien, Taiwan, ROC.
Kuo
Hsien-Shou
HS
Tsai
Hsin-Da
HD
Peng
Yi-Chen
YC
Lin
Yuh-Ling
YL
D018931:Antineoplastic Agents, Hormonal / Q000633:toxicity; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity; D017209:Apoptosis / Q000235:genetics*; D001388:Aziridines / Q000633:toxicity*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D005260:Female; D016158:Genes, p53; D006801:Humans; D009285:Naphthoquinones / Q000633:toxicity*; D017239:Paclitaxel / Q000633:toxicity; D013629:Tamoxifen / Q000633:toxicity; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000502:physiology
C507359:2-aziridin-1-yl-3-((2-((2-(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)ethoxy)ethyl)thio)naphthoquinone; D018931:Antineoplastic Agents, Hormonal; D000972:Antineoplastic Agents, Phytogenic; D001388:Aziridines; D009285:Naphthoquinones; D016159:Tumor Suppressor Protein p53; D013629:Tamoxifen; D017239:Paclitaxel