The Effect Of Doxycycline On SKOV3 And Its Stem Like Cells (SKOV3/CSC) In Vitro

Objectives: Study of the effect of Doxycycline on SKOV3 and its stem like cells( SKOV3/CSC) in vitro and explore the anti-tumor mechanism of doxycycline.Methods: 1 SKOV3 cells and its cisplatin resistance cell SKOV3 / CDDP cells were cultured in vitro, observed cells morphological by inverted microscope.The SKOV3 cells were cultured in cancer stem cell specific medium to enrich the SKOV3/CSC cells. Flow cytometric to detect the expression of CD133 and ALDH(Aldefluor assay) of SKOV3 and SKOV3/CSC cells. MTS assay to test the inhibition ratio of SKOV3,SKOV3/CDDP and SKOV3/CSC in different concentration of doxycycline(0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml).Also use MTS assay to test the influence of doxycycline on the sensibility of SKOV3/CDDP and SKOV3/CSC cells to cis-platinum. 2 Flow cytometric to test the influence of different concentration of doxycycline(0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml) on apoptosis and the mitochondrial membrane potential in SKOV3 and SKOV3/CSC cells.And use the electron microscope to observe the influence of 20μg/ml doxycycline on the ultrastructure of SKOV3 and SKOV3/CSC cells. 3 Western-Blotting test the influence of different concentration of doxycycline( 0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml) on the relative expression of Caspase-3,Caspase-9 and cytochrome( in cytoplasm). 4 The influence of different concentration of doxycycline on invasion and migration potential of SKOV3(0μg/ml,5μg/ml,10μg/ml)and SKOV3/CSC(0μg/ml,5μg/ml)were assayed using Transwells.Result:1 Flow cytometric analysis showed that the positive rate of CD133 in SKOV3 and the first, third, fifth, seventh, ninth generation of SKOV3 / CSC cells were(0.99±0.02)%,(1.11±0.06)%,(1.34±0.05)%,(1.26±0.14)%,(1.32±0.07)%,(1.93±0.14)%.With the increase of subculture the positive rate of CD133 were not significantly increased.And the ALDH positive rate were(0.35±0.01)%,(0.79±0.02)%,(33.30±0.16)%,(67.65±0.44)%,(90.24±0.16)%,(91.66±0.64)%. With the increase of subculture the positive rate of ALDH increased significantly. 2 MTS analysis showed that doxycycline has an inhibitory effect on SKOV3,SKOV3/CDDP and SKOV3/CSC,and it also can increase sensitivity to cisplatin. 2.1 Doxycycline has an inhibitory effect on SKOV3. SKOV3 cells treated with 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h,the inhibition ratio were(-4.39±2.99)%,(-4.32±3.35)%,(11.76±1.73)%,(36.24±2.01)%( P=0.232>0.05,P=0.279>0.05,P=0.001<0.05,P<0.0001),for 48 h the inhibition ratio were(-3.71±2.20) %,( 0.94±0.92) %,( 3.18±1.20) %,(47.74±2.35)%(P=0.1487>0.05,P=0.4499>0.05,P<0.0001,P<0.0001). 2.2 Doxycycline has an inhibitory effect on SKOV3/CDDP. SKOV3/CDDP cells treated with 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h,the inhibition ratio were(2.38±1.65)%,(11.63±1.23)%,(36.74±1.38)%,(50.54±0.85)%。(P=0.3219>0.05,P=0.0004>0.05,P<0.0001,P<0.0001),for 48 h the inhibition ratio were(5.57±0.30)%,(18.75±0.25)%,(40.63±0.73)%,(56.77±0.49)%(P<0.0001,P<0.0001,P<0.0001,P<0.0001). 2.3 Doxycycline has an inhibitory effect on SKOV3/CSC. SKOV3/CSC cells treated with 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h,the inhibition ratio were(2.75±0.52)%,(17.28±2.30)%,(26.02±1.70)%,(53.88±2.12)%(P=0.0914>0.05,P=0.0002<0.05,P<0.0001,P<0.0001),for 48 h the inhibition ratio were(9.68±2.95)%,(23.84±2.87)%,(49.20±2.98)%,(70.58±1.22)%(P=0.0373<0.05,P<0.0001,P<0.0001,P<0.0001). 2.4 SKOV3/CDDP and SKOV3/CSC cells were more sensitive then SKOV3 cells. The IC50 of SKOV3/CDDP,SKOV3/CSC cells were 36.05±0.05)μg/ml,(36.85±0.13)μg/ml, and The IC50 of SKOV3 cells was(50.32±0.52)μg/ml(P<0.0001,P<0.0001). 2.5 Doxycycline sensitize SKOV3/CDDP and SKOV3/CSC cells to cisplatin. The IC50 of cisplatin of SKOV3/CDDP cells in the presence of doxycycline(0μg/ml,5μg/ml) were(16.81±0.20)μg/ml and(11.12±0.09)μg/ml(P<0.0001). The IC50 of cisplatin of SKOV3/CSC cells in the presence of doxycycline(0μg/ml,5μg/ml) were(17.40±0.03)μg/ml and(9.36±0.10)μg/ml(P<0.0001). 3 Flow cytometric analysis showed that doxycycline can induce SKOV3 and SKOV3/CSC cells apoptosis, mitochondrial membrane potential decreased,and electron microscope results show cell ultrastructure change. 3.1 The apoptosis ratio of SKOV3 cells after treatment of 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h were(3.89±0.07)%,( 3.95±0.06) %,( 4.03±0.07) %,( 5.53±0.06) %.(P=0.1266>0.05,P=0.0546>0.05,P<0.027,P<0.0001). 3.2 The apoptosis ratio of SKOV3/CSC cells after treatment of 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h were(3.45±0.14)%,( 8.14±0.21) %,( 10.27±0.18) %,( 19.55±0.32) %.(P=0.7724>0.05,P<0.0001,P<0.0001,P<0.0001). 3.3 The mitochondrial membrane potential decrease rate of SKOV3 cells after treatment of 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h were( 3.81±6.45) %,( 21.53±3.72) %,( 50.54±4.72) %,( 71.67±8.73)%,(P=0.6611>0.05,P=0.0242<0.05,P<0.0001,P<0.0001). 3.4 The mitochondrial membrane potential decrease rate of SKOV3/CSC cells after treatment of 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h were(15.24±2.83)%,(36.86±3.46)%,(49.57±3.89)%,(96.58±3.42)%(P=0.0085 <0.05,P=0.0006<0.05,P=0.0003<0.05,P<0.0001). 3.5 Transmission electron microscope found that the mitochondria of SKOV3 cells were swelling mildly, crest fractured, part of the membrane fusion and ambiguous after the treatment of 20μg/ml doxycycline.And the mitochondria of SKOV3/CSC cells were swelling seriously, mitochondrial cavitation, medullary mitochondria.Other organelles has no obvious change. 4 Western-Blotting analysis showed that after the treatment of 5μg/ml,10μg/ml,20μg/ml,40μg/ml doxycycline for 24 h the relative expression of C aspase-9 were 0.39±0.00,0.54±0.01,0.73±0.01,0.99±0.00,(P<0.0001,P<0.0001,P<0.0001,P<0.0001);the relative expression of Caspase-3 were 0.55±0.00,0.69±0.01,0.75±0.00,0.94±0.00,(P<0.0001,P<0.0001,P<0.0001,P<0.0001);and the relative expression of cytochrome C in cytoplasm were 0.40±0.00,0.52±0.01,0.74±0.001,0.84±0.00,(P=0.0025<0.05,P=0.0002<0.05,P<0.0001,P<0.0001). 5 The Transwell assay showed that after the treatment of 5μg/ml,10μg/ml doxycycline for 24 h the migration of SKOV3 cells were decreased(46.08±4.82)%,(74.41±1.30)%(P<0.0001,P<0.0001),and the invasion wer e decreased(4.31±2.36)%,(40.57±2.79)%(P=0.5103>0.05,P=0.0002<0.05).And after the treatment of 5μg/ml doxycycline for 24 h the migratio n of SKOV3/CSC cells were decreased(29.74±1.74)%,(P=0.001<0.05),and the invasion were decreased(34.67±3.16)%,(P<0.0001).Conclusion: 1 Doxycycline has an inhibitory effect on SKOV3,SKOV3/CDDP and SKOV3/CSC,and it also can increase sensitivity to cisplatin. 2 Doxycycline can induce SKOV3/CSC cells apoptosis through the mitochondrial pathways. 3 Doxycycline can inhibite the migration and invasion of SKOV3 and SKOV3/CDDP cells. 4 Serum-free culture method can be a effective mean to enriched ALDH + SKOV3 / CSC cells. 5 CD133 may not applicable to identify SKOV3/CSC cells that enriched by Serum-free culture method. 6 Mitochondria could become a new targets for the eradication of ovarian cancer, doxycycline, as a kind of mitochondrial protein synthesis inhibitors, may be in a new effective anti-tumor drugs, finally provides new hope for us to overcome ovarian cancer.