Effect And Mechanism Of Hematoporphyrin Activated By Ultrasound On Anoikis Resistance In HepG2 Hepatocarcinoma Cells

Objective: To study the anoikis resistance in HepG2 cells and the effect of hematoporphyrin activated by ultrasound on its anoikis resistance and research the potential mechanism.Methods: (1) The growth of the HepG2 cells and HUVEC cells in the condition of adherence loss was observed by cell colony formation in soft agar experimemt. The anoikis of the HepG2 cells and HUVEC cells was detected by agarose gel electrophoresis and flow cytometry. (2) The HepG2 cells were randomly divided into four groups as follows: the cells in hematoporphyrin group incubated in culture solution containing 40μl hematoporphyrin (1.5mg/ml)for 30s away from light, those in ultrasound group irradiated by 1.0MHz, 1.5W/cm2 ultrasound for 30s, those in hematoporphyrin and ultrasound group incubated in culture solution containing hematoporphyrin in the same dose and then irradiated by ultrasound in the same intensity for 30s, and others as the control group. After treatment the growth of the HepG2 cells in the condition of adherence loss was observed by cell colony formation in soft agar experimemt. The anoikis of the HepG2 cells was detected by agarose gel electrophoresis and flow cytometry. (3) There was a hematoporphyrin, histidine and ultrasound group except for four groups mentioned above, and the HepG2 cells in that group were incubated in culture solution containing 40 ul hematoporphyrin and 31μl histidine for 30s away from light and irradiated by 1.0MHz, 1.5W/cm~2 ultrasound for 30s. After treatment the relative loss of DPBF was evaluated by ultraviolet-visible spectrophotometer, the apoptotic rate was counted by flow cytometry, and the expression of OPN was analysised by RT-PCR and Western blot.Results: (1) The inverted microscope exhibited that the HepG2 cells grew in the condition of adherence loss and formed cell colonies in soft agar. However the HUVEC cells didn't form cell colonies, the DNA ladder appeared and flow cytometry detected that the apoptosis rate of the HUVEC cells was lower than that of the HepG2 cells. (2) In hematoporphyrin and ultrasound group the cell colonies of the HepG2 cells in soft agar were less than other groups, the DNA ladder was observed,and the apoptosis rate was higher than other groups. (3) In hematoporphyrin and ultrasound group ultraviolet-visible spectrophotometer detected that the relative loss of DPBF was more than other groups, flow cytometry detected that the apoptosis rate was higher, and RT-PCR and Western blot detected that the expression of OPN was lower.Conlusion: (1) The HepG2 cells can survive in the condition of isolation, and it has the potential of anoikis resistance.(2) Hematoporphyrin activated by ultrasound can inhibit the anoikis resistance in HepG2 cells.(3) Singlet oxygen decreases the expression of OPN may be the mechanism through which hematoporphyrin activated by ultrasound inhibits the anoikis resistance in HepG2 cells.