Doxycycline Inhibits The Growth And Induces The Apoptosis Of Human Hepatoma Cell HepG₂ In Vitro

ObjectiveSince oncogenesis and development in hepatocarcinoma correlate closely with apoptosis of cancer cells,the induction of apoptosis in hepatoma cells has played a more and more important role in therapy of hepatocarcinoma at present.Doxycycline belongs to antibiotic family of tetracycline with effictiveness against several diseases caused by microbial infection,which is also found have the anti-tumor properities in the case of colon,breast,pancreatic and prostatic cancer.In this study,we observed the effect of doxycycline on cell growth inhibition and apoptosis of human hepatoma cell HepG2 in vitro.Then we explored the possible mechanism by assessing the protein and mRNA expression of FasL,Fas and MMP-2.Methods1.HepG₂ cells were treated with doxycycline in different concentrations (5,10,20,30 and 50mg/L)and time(24,48 and 72hours)in vitro.Invert microscope was used to observe the changes of cell appearance,and to take photos.2.The growth inhibiton ratio of HepG₂ cells was detected by MTT assay.The control group were only treated by RPMI1640 culture medium with calf serum for 24, 48 and 72 hours respectively;The doxycycline group were treated by 5,10,20,30 and 50mg/L doxycycline for 24,48 and 72 hours respectively.3.The apoptosis rate of HepG₂ cells was identified by TUNEL assay.The control group were only treated by RPMI1640 culture medium with calf serum for 24, 48 and 72 hours respectively;The doxycycline group were treated by 5 and 20mg/l doxycycline for 24,48 and 72 hours respectively. 4.The cell growth cycles of HepG₂ cells were examined by PI staining flow cytometry.HepG₂ cells were divided into three groups:the control group was just treated by RPMI1640 culture medium with calf serum for 24,48 and 72 hours respectively;the doxycycline groups were treated by 5 and 20mg/l doxycycline for 24,48 and 72 hours.5.The protein expression of FasL,Fas and MMP-2 in HepG₂ cells was evaluated by Strept avidin-peroxidase conjugated method(SP)of immunocytochemistry.HepG₂ cells were divided into two groups:the control group was just treated by RPMI1640 culture medium with calf serum for 48 hours;the second group was treated by 20mg/L doxycycline for 48h.6.The mRNA expression of FasL,Fas and MMP-2 in HepG₂ cells was assessed by Reverse Transcription Polymerase Chain Reaction(RT-PCR).HepG₂ cells were divided into three groups:the control group was just treated by RPMI1640 culture medium with calf serum for 48 hours;the doxycycline groups were treated by 5 and 20mg/l doxycycline for 48h.Results1.Cell changes were observed in an inverted microscope:cells in the control group grew well,the cells extended,transparent,formed irregular angular forms,and close together.The doxycycline group cells decreased in number,bubbles formed in the cytoplasm,their areas decreased,they became round,the distance between them increased and a large number of suspended apoptotic cells could be seen with the naked eye.2.Treated with doxycycline at different concentrations(5,10,20,30,50mg/L)for 48 and 72 hours,The differences of the proliferative inhibition rates between the control groups and doxycycline groups were statisticaly significant(P＜0.05)and there was a tendency of dose-time dependence,whereas those treated for 24 hours showed no difference(P＞0.05).3.Treatment with doxycycline enhanced apoptosis in HepG₂ cells.The apoptosis rate showed a significant increase(P＜0.01)after treatment with doxycycline 5 and 20mg/L for 48 and 72 hours compared with the control group.The difference was insignificant after treatment for 24h.4.Flow cytometer analysis of HepG₂ cell cycles showed the figure of doxycycline group was much larger on G₁ phase and much lower on S phase compared with the control group.That is to say G₁-S phase block and S phase inhibition is the major alteration of the cell cycle.5.Incubated HepG₂ cells with doxycycline 20mg/L for 48 hours,the protein expression of FasL was up-regulated and MMP-2 was down-regulated significantly comprared with those of control group(p＜0.01),while the difference of Fas expression was insignificant(p＞0.05).6.After exposure to doxycycline 20mg/L,the mRNA expression of FasL increased significantly when compared with the control group(P＜0.01),while the mRNA expression of MMP-2 decreased significantly.No statistically significant effect was observed on Fas mRNA expression in HepG₂ cells.Conclusion1.Doxycycline can inhibit cell growth and induce apoptosis of HepG₂ significantly.2.Doxycycline has the ability of G₁-S phase block and S phase inhibition on HepG2 cells.3.The mechanism of doxycycline induced apoptosis might be associated with down-regulating expression of MMP-2 and up-regulating expression of FasL on HepG₂ cells,which activated the Fas/Fasl signal transduction pathway to mediate apoptosis.