Analysis Of The Effects Of Artemisinin Derivatives On The Biological Behavior Of Cervical Cancer Cells And On The Angiogenisis Of Human Umbilical Vein Endothelial Cells In Vitro

Objective:1. To investigate the effect and the molecular mechanism of artemisinin derivatives (dihydroartemisinin and artesunate, DHA and ART) on the proliferation、apoptosis of cervical cancer cells (Hela and Caski) in vitro.2. To investigate the effect and the molecular mechanism of DHA and ART on the invasion of cervical cancer cells (Hela and Caski) in vitro.3. To investigate the effect of DHA and ART on the proliferation and angiogenesis of human umbilical vein endothelial cell in vitro.4. To investigate the effect of DHA and ART on the proliferation of cisplatin-resistant cervical cancer cell (Siha) in vitro.Methods:1. Hela and Caski cell lines were cultured respectively in Dulbecco’s Modified Eagle’s Medium (DMEM)/high-glucose medium and Roswell Park Memorial Institute-1640medium supplemented with10%fetal bovine serum,100mg/ml penicillin and100mg/ml streptomycin at5%CO2and37℃. Cervical cancer cells were exposed to indicated concentrations (0μmol/L～62.5μmol/L) of DHA, ART and DDP for different hours (6-72h), and the MTT assay was used to observe the effect of DHA, ART and DDP on the proliferation of cervical cancer cells in vitro. And the flow cytometry was used to verify their effect on the apoptosis of cervical cancer cells in vitro. Then the phosphorylation level of MAPKs (mitogen-activated protein kinases) of cervical cancer cells was detected by Western blot.2. The supernatant which were collected after the treatment of DHA and ART were examined by using enzyme-linked immunosorbent assay to verify the effect of DHA and ART on the secreted proteins such as VEGF, TSP-1, IP-10, MMPs/TIMPs which were related to the angiogenesis and invasion of cervical cancer cells. Then the mRNA of these molecules was detected by using polymerase chain reaction (PCR). And Western blot was used to test the phosphorylation level of FAK (Focal Adhesion Kinase) of cervical cancer cells. Then the effects of DHA and ART on the invasive ability of cervical cancer cells were observed by using Matrigel coated transmembrane cell model.3. Human umbilical vein endothelial cell was cultured in M199medium supplemented with10%fetal bovine serum,100mg/ml penicillin,100mg/ml streptomycin and ECGF at5%CO2and37℃and HUVEC was exposed to indicated concentrations (0μmol/L～125μmol/L) of DHA and ART for different hours(6-72h), and the MTT assay was used to observe the effect of DHA and ART on the proliferation of cervical cancer cells in vitro. And the tube-like structure formation models was applied to observe the effect of DHA on the tube-like structure forming ability of HUVEC in vitro.4. Siha (cisplatin induced multidrug resistant cell line) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/high-glucose medium supplemented with10%fetal bovine serum,100mg/ml penicillin and100mg/ml streptomycin at5%CO2and37℃. Siha was exposed to indicated concentrations (0μmol/L-125μmol/L) of DHA, ART for different hours (6-72h), and the MTT assay was used to observe the effect of DHA and ART on the proliferation of Siha in vitro.Results:1. DHA and ART significantly inhibited the growth of cervical cancer cells (Hela and Caski) in vitro and the inhibition was dependent on the drug concentration and action time. The IC50of Hela cell lines by DHA and ART for48h were8.9±1.1μmol/L and10.5±1.2μmol/L respectively, and the IC50of Caski cell lines by DHA and ART for72h werel0.3±1.0u mol/L and13.3±1.4μmol/L respectively.2. Compared with the control group, DHA reduced the phosphorylated ERK1/2levels of Hela and Caski by70.5±2.6%(p<0.05) and68.8+2.2%(p<0.05) while ART reduced the phosphorylated ERK1/2levels of Hela and Caski by65.8±2.9%(p<0.05) and56.7±2.4%(p<0.05)3. DHA and ART can promote the apoptosis of Hela and Caski in vitro. And DHA increases the apoptosis of Hela and Caski after treatment with12h to51.0±3.6%(p <0.05) and24.6±2.5%(p<0.05) respectively, while ART increases the apoptosis of Hela and Caski after treatment with12h to38.4±3.2%(p<0.05) and19.3±2.7%(p<0.05) respectively. 4. Compared with the control group DHA increased the phosphorylated p38levels of Hela and Caski by57.3±2.4%(p<0.05) and54.8±3.1%(p<0.05) while ART increased the phosphorylated p38levels of Hela and Caski by47.0±3.0%(p<0.05) and43.8±2.8%(p<0.05)5. DHA and ART were able to inhibite the invasion ability of Hela and Caski in vitro. DHA reduced the invasion capacity of Hela and Caski by76.2±2.6%(p<0.05) and67.2±2.9%(p<0.05), while ART reduced the invasion capacity of Hela and Caski by68.1±2.7%(p<0.05) and59.3±2.8%(p<0.05)6. Compared with the control group, DHA reduced the phosphorylated FAK levels of Hela and Caski by66.8±2.2%(p<0.05) and54.6±2.7%(p<0.05) while ART reduced the phosphorylated FAK levels of Hela and Caski by48.9±2.6%(p<0.05) and42.1±2.3%(p<0.05)7. DHA and ART significantly inhibited the growth of human umbilical vein cell in vitro. The IC50of HUVEC by DHA and ART for72h were11.4±1.4μmol/L and17.1±1.1μmol/L respectively.8. And compared with the control group,62.5u mol/L DHA made tube-like structure formation decreased by62.3%whose effect is equivalent to the10"9mol/L paclitaxel.9. DHA and ART also inhibited the growth of Siha cell line which was cisplatin induced multidrug resistant cell line in vitro. The IC50of Siha by DHA and ART for72h were36.1±1.7μmol/L and43.0±1.5u mol/L respectively.Conclusion:1. For the cervical cancer cell lines (Hela and CaSki), DHA and ART can inhibite their growth ability, and the inhibition might be related to the down-regulating phosphorylation level of ERK1/2.2. DHA and ART can promote the apoptosis of Hela and Caski in vitro. And the pro-apoptosis was associated with up-regulating phosphorylation level of P38.3. DHA and ART were able to inhibite the invasion ability of Hela and Caski in vitro. And the suppressed invasion activity was related to down-regulating the phosphorylation level of FAK.4. DHA and ART can inhibited the proliferation of human umbilical vein cell and its tube-like structure formation ability in vitro.5. DHA and ART can inhibite the proliferation of Siha which is cisplatin induced multidrug resistant cell line in vitro.