Ultrasound Irradiating Hydroxycamptothecin-Loaded Microbubbles For Proliferation Inhibition Of Human Hepatoma SMMC-7721 Cells

Objective: To investigate the proliferation inhibition effect of hydroxyl camptothecin (HCPT) -carrying microbubbles on human hepatoma SMMC-7721 cells using technique of ultrasound mediated drug release.Methods: The SMMC-7721 cells were randomly divided into six groups as follows:Ⅰcontrol group,Ⅱblank liposome microbubbles plus ultrasound group,ⅢHCPT-loaded microbubbles group,ⅣHCPT group,ⅤHCPT plus ultrasound group, andⅥHCPT-loaded microbubbles plus ultrasound group.(1)The cell growth inhibitory effect of each group was observed by MTT assay.(2)Cell cycle distribution of each group was analyzed using Flow Cytomertry (FCM). (3) Cell apoptosis at early term of each group was ascertained by Annexin V-Fitc/PI method. (4) Cell apoptosis at advanced term of each group was detecteded by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL). (5) The expression of Bcl-2 and Bax protein of SMMC-7721 cells of each group were analyzed by immunocytochemistry staining technique.Results: (1) Each factor could inhibit the proliferation of SMMC-7721 cells in a time-dependent manner. The cell inhibitory rate in the group of combining HCPT-carrying microbubbles and ultrasound at 12 h, 48 h, 72 h respectively was (49.56±1.57)%, (80.01±1.52)%, (91.41±0.64)%, which was significantly higher than other groups ( P <0.05). (2) The results of FCM showed that the distributional percentage of cell cycle in HCPT-loaded microbubbles group, HCPT group, HCPT plus ultrasound group, and HCPT-loaded microbubbles plus ultrasound group changed 24h after treatment ,which mainly was an increase in the proportion of G0/G1 phase and a decrease in the proportion of S phase in cell cycle. The increase in the proportion of G0/G1 phase was the most significant in the group of combining HCPT-carrying microbubbles and ultrasound ( P <0.05). The change of cell cycle in blank liposome microbubbles plus ultrasound group mainly was an increase in the proportion of G2/M phase. (3) The apoptosis rate at early term in control group, blank liposome microbubbles plus ultrasound group, HCPT-loaded microbubbles group, HCPT group, HCPT plus ultrasound group, and HCPT-loaded microbubbles plus ultrasound group was found to reach (2.58±0.20)%, (4.57±0.59)%, (4.60±0.86)%, (6.21±0.87)%, (8.22±1.03)%, (12.18±1.38)% respectively by Annexin V-Fitc/PI assay. The result of HCPT-loaded microbubbles plus ultrasound group was significantly different with other groups ( P <0.05). (4) The apoptosis rate at advanced term in six groups was found to reach (2.32±1.00)%, (6.57±1.76)%, (7.90±1.06)%, (16.44±0.65)%, (23.64±0.63)%,(34.25±1.83)% respectively by TUNEL method. The result of HCPT-loaded microbubbles plus ultrasound group was significantly different with other groups ( P <0.05). (5) The results of immunocytochemistry staining assay showed five factors had an impact on the protein expression of Bcl-2 and bax of SMMC-7721 cells. But in HCPT-loaded microbubbles plus ultrasound group, the expression of Bcl-2 was significantly lower than that in other groups, the expression of Bax was significantly higher than that in other groups, and the ratio of Bcl-2 to bax was decrease significantly( P <0.05).Conclusion: The method of ultrasound irradiating HCPT-carrying microbubbles significantly inhibited the human hepatoma SMMC-7721 cells proliferation. Cell cycle arrest and apoptosis induction by the decrease in the ratio of Bcl-2 to Bax might be responsible for cells growth inhibition.