| Berberine inhibits the growth of various tumor cell types, including hepatocellular carcinoma, colon cancer, lung cancer, oral cancer, bladder cancer, breast cancer, and prostate cancer. Berberine has a significant superiority with its low cytotoxic effect on normal human cells. Although stable in the gastrointestinal tract, berberine is poorly absorbed and low bioavailability in the intestine, resulting in insufficient plasma concentrations and low efficacy. In order to reduce the toxicity, enhance the liposolubility and bioavailability of berberine, berberine-bile acids analogues were synthesized by nucleophilic substitution, amidatio and esterification at C-9of berberine in order to evaluate the influence of the different bile acid substitute group on different position of berberine. We found2,3-methenedioxy-9-O-(3’a,7’a-dihydroxy-5’β-cholan-24’-propy-lester) berberine (B4), conjugated with chendeoxycholic acid by ester linkers, exerted higher bioavailability (more4.7times than berberine) and better antimicrobial activity. B4showed anticancer activity against hepatoma cancer cells SMMC-7721and BEL-7402, human gastric cancer cells SGC-7901and colon cancer cells HCT116. In this paper, we further studied the underlying molecular mechanisms of apoptosis of B4towards SMMC-7721cells by flow cytometry, LSCM and Western Blot. The study results were as follows:The anti-proliferative activity and cytotoxic effect on normal human cells of B4was analyzed by the MTT assay. It was found that B4elicited a greater inhibitory effect than berberine in a dose-dependent manner and time-dependent manner; berberine exhibited a greater cvtotoxic effect on normal human liver cells (HL-7702) at all doses>0.4μM compared to B4. In all subsequent experiments,2μM B4was the highest concentration used since it inhibited proliferation of normal cells by<20%.The morphological changes were observed with laser scanning confocal microscopy (LSCM). The results showed that SMMC-7721cells treated with2μM B4after48h displayed morphology changes such as cell density decreased, the numbers of floating cells increased, nuclear condensation, marginalization of the fragmented nuclei towards the membrane, and apoptotic bodies were formed.The cell cycle distribution and apoptosis were analyzed by flow cytometry. After SMMC-7721cells treament with B4, the percent of cells at S phase was increased and the percent of cells at G0/G1phase and G2/M phase were reduced in a dose-dependent manner. B4induced the accumulation of SMMC-7721cells at S phase. Meanwhile, the percentage of total apoptotic cells increased significantly in a concentration-dependent manner, and B4elicited a greater induced-apoptosis effect on hepatoma cells compared to berberine. The changes of intracellular Ca2+, mitochondrial membrane potential (△ψm) and reactive oxygen species (ROS) were analyzed by flow cytometry respectively. The study results showed that the concentrations of intracellular Ca2+and ROS were increased and△ψm was reduced in a dose-dependent manner with increasing concentration of B4. To further study the role of ROS in the B4-induced apoptosis, SMMC-7721cells were pretreated with the antioxidant N-acetylcysteine (NAC) for1h before incubated B4for48h. Pretreatment with NAC, significantly blocked the B4-mediated rise in ROS, decreased the Ca2+level and△ψm level to that of control. Our results suggest that ROS production plays an important role in B4-induced apoptosis.Western blot analyzed the relative proteins of B4-induced apoptosis. The study results showed that B4caused a rise in the cleavage fragment of the caspase-3substrate poly (ADP-ribose) polymerase (PARP) and a dose-dependent decrease XIAP protein and caspases. Additionlly, we found that AIF translocated from mitochondria to the nucleus in a dose-dependent manner. B4induced apoptosis of SMMC-7721cells in the caspase-dependent and caspase-independent pathway. However, pretreatment with the anti-oxidant NAC prevented B4-induced apoptosis by inhibiting ROS generation. NAC offered significant protection against B4-induced mitochondrial membrane depolarization, PARP cleavage and AIF translocation. Together, these data suggest that ROS generation is an early event in the process of B4-induced apoptosis.In this paper, B4had a significant anti-tumor activity in vitro. We had provided B4played the anti-tumor effect by inducing the SMMC-7721cells apoptosis through the morphology, cell cycle distribution and apoptotic experments, and futher studied the apoptosis mechanism on the molecular level. |
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