The Apoptosis Mechanism Of Camptothecin-20(s)-O-[N-(3’α,12’α-dihydroxy-24’-carbonyl-5’β-Cholan)]-lysine Towards Hepatocellular Carcinoma SMMC-7721Cells

In this paper, we studied the apoptosis mechanism that the new camptothecin (CPT)-bile acid analogue Camptothecin-20(s)-O-[N-(3’α,12’α-dihydroxy-24’-carbonyl-5’β-cholan)]-lysine (B2) induced hepatocellular carcinoma SMMC-7721cells.In order to reduce the toxicity, enhance the targeting ability and bioavailability of CPT and its derivatives, we evaluated the influence of the different bile acid substitute group on different position of CPT. Our previous study had shown that B2was the best effective in vitro and good anti-tumor activity at lower concentration in vivo. In this paper we further researched the underlying mechanism of apoptosis of B2towards SMMC-7721cells by flow cytometry, LSCM and protein electrophoresis. The results were as follows:The anti-proliferative activity of B2was analyzed by the MTT assay. It was found that B2could inhibite proliferation of several tumor cell lines including hepatocellular carcinoma SMMC-7721cells and BEL-7402cells and human colon cancer HCT-116cells, the SMMC-7721cell line was the most sensitive cell line compared to the positive control group. The growth of SMMC-7721cells was significantly inhibited in a dose-and time-dependent manner by B2. When SMMC-7721cells were treated with0.4nM B2after48h,41.26%±2.82%of cells were killed.The morphological changes were observed under laser scanning confocal microscopy (LSCM), the results showed that the cells treated with0.5nM B2after48h displayed morphology changes such as 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. The cell cycle distribution was changed after the cells treated with the different concentrations of B2. With the increasing concentrations of B2, the percentages of cells in S phase and G2M phase were increasing, while the percentage of cells in G1phase was decreasing. The results indicated the cell cycle was arrested in S phase and G2M phase. Meanwhile the apoptoic results showed that the rate of apoptosis was increased in a concentration-dependent manner after the cells treated by B2.The changes of intracellular calcium, mitochondrial membrane potential (△(?) m) and reactive oxygen species (ROS) were analyzed by flow cytometry respectively using Fluo-3AM, Rh123and fluorescent probes DCFH-DA. The results showed that the concentrations of intracellular calcium and ROS were increased and the mitrchondrial membrane potential was reduced in a dose-dependent manner after the SMMC-7721cells treated with B2.The changes of caspase-3and caspase-9were analyzed by spectrophotometric method using microplate reader. The results showed that B2induced SMMC-7721cells apoptosis by activating caspase-3and caspase-9.The apoptosis-related proteins were analyzed by western blot. In our study, B2induced SMMC-7721cells apoptosis, which was accompanied by down-regulation of Bcl-2protein and up-regulation of Bax protein.In this paper, B2had a significant anti-tumor activity in vitro. We had provided B2played 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.