Inhibition Activity And Apoptosis Mechanism Of Two Novel Asymmetric Curcumin Analogues To Human Breast Cancer MCF-7 Cells

In this paper, we studied the apoptosis mechanism that the two novel asymmetric curcumin analogues synthesized in our laboratory:(1E,4E)-1-(4-hydroxy-3,5-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (F3), and (1 E,4E)-1-(3,4-dimethoxyphenyl)-5-(2-hydroxy-4-methoxypheny l)penta-1,4-dien-3-one(LE) induced human breast cancer MCF-7 cells.The cytotoxicity of F3 and LE were investigated by MTT assays. It was found that both F3 and LE could inhibit proliferation of several tumor cell lines including hepatocellular carcinoma SMMC-7721 cells and human breast carcinoma MCF-7 cells and human prostate carcinoma PC-3 cells, the MCF-7 cell line was the most sensitive cell line. Both F3 and LE exerted higher killing ability than Curcumin. The morphological analysis using a laser scanning confocal microscope(LSCM) after the cells were dyed by acridine orange solute (AO) Results showed that the cells teated with F3 or LE displayed typical apoptotic features such as nuclear and cytoplasmic condensation, chromatin fragmentation, marginalization of the fragmented nuclei towards the membrane, and apoptotic bodies.Futher cell cycle distribution analysis, mitochondrial transmembrane potentials (Δφm), and apoptosis assays via a flow cytometry (FCM). The cell cycle distribution was changed after the cells treated with the different concentrations of F3 or LE. The proportion of cells at S phase increased slightly and those in G0/G1 phase decreased in a dose-dependent manner in MCF-7 cells exposed to F3. An apparent increase in the proportion in G2/M phase was also observed. Treatment with LE also significantly increased the proportion of cells in S phase and reduced the number in G0/G1 phase in a dose-dependent manner, and slightly increased the proportion in G2/M phase. The mitochondrial membrane potential was ruduced in a dose-dependent manner and the percentage of apoptotic cells (annexin V+) increased significantly with increasing concentrations of both F3 and LE. Taken together, these findings indicated that F3 and LE reduced MCF-7 cell viability by inducing apoptosis.In order to realise the apoptosis mechanism of F3 and LE towards MCF-7 cells we used western blot assays to determine the expressions of cytochrome c, apoptosis-related factors and p38MAPK at protein level.Western blot results showed that the curcumin analogues inhibited Bcl-2 and induced Bax and Bak expressions and led to release of cytochrome c and AIF from the mitochondria to the cytosol. F3 and LE activated the caspase-9 and -3 in the cytosol but procaspase3 were reduced by F3 slightly. Cleavaged of PARP in the nucleus and decreases the level of XIAP were also be found. In addition, treatment with either F3 or LE increased the amount of phosphorylated p38 in a concentration dependent manner, without altering the total p38 protein level. In contrast, F3 and LE did not affect the levels of phospho-JNK. We further investigated the role of p38MAPK by employing a pharmacological inhibitor of p38MAPK (SB203580). This inhibitor attenuated both F3-induced and LE-induced apoptosis.Our data suggested that F3 and LE induced apoptosis in MCF-7 cells via mitochondrial-related apoptotic pathway and p38MAPK signaling contributed to this process.