Effects Of Low-dose Metronomic Chemotherapy On Vasculogenic Mimicry Of Ovarian Cancer Cell SKOV3 And The Possible Mechanisms

Objective:Vasculogenic mimicry(VM) is a highly aggressive tumor cells for self blood supply microcirculation structure. To explore the effects of low-dose metronomic chemotherapy(LDM) and maximum tolerated-dose chemotherapy(MTD) on VM of ovarian cancer cell SKOV3 and the possible mechanisms. Obtained the primary tumor cells through injecting cisplatin by different chemotherapy schedules(LDM and MTD) in the nude mice bearing SKOV3 ovarian cancer xenografts and compared the VM formation ability, proliferate ability, and invasion ability of the primary cells. Analysed the expression level of CD133 and ALDH1 in the primary cells by Western-blot and observed the VM formation differences of the CD133+ALDH1+ cells and CD133-ALDH1- cells that insolted by fluorescence-activated cell sorting(FACS).Methods:1 To establish the nude mice xenograft tumor models, divide into three groups, Adopt different chemotherapy schedules of cisplatin.For subcutaneous(s.c.) tumor formation, SKOV3 cells(1×107) were suspended in 0.2 ml phosphate-buffered saline(PBS) and s.c. injected into the right thigh of each nude mouse. Approximately two weeks later, the mice were randomly divided into three groups as follows: LDM group(intraperitoneal injection of 1mg/kg cisplatin daily for eighteen days), MTD group(intraperitoneal injection of 3mg/kg cisplatin once every three days for six cycles) and control group nude mice were treated with the equal saline solution.2 Made and Cultured primary tumor cellsIn 3-7 days after chemotherapy finished,the turmors were dislodged and the single cell suspension was made by a series of steps : enzymatic digestion, milling-in, filtration, centrifugate, scrubbing and resuspend. The three groups of primary tumor cells were obtained by culture the single cell suspension for 72 hours finailly.3 Set up three-dimensional cell culture model, Observed the VM formation ability of three group primary tumor cellsThe Single primary tumor cell suspension was cultured in 3DCC model. We observed the form conditions of VM by inverted microscope and counted the vasculogenic mimicry density(VMD). The experiment was repeated for 3 times and recorded by photoes.4 MTT was used to detect the proliferate ability of SKOV3 primary cells though different chemotherapy schedules of cisplatin.5 The invasion ability of each group cells was evaluated by the Transwell chamber test in vitro.6 The expression level of CD133 and ALDH1 in primary tumor cells was detected with Western-blotting method.7 Isolation CD133+ALDH1+ cells and CD133-ALDH1- cells by fluorescence-activated cell sorting(FACS)The primary cells of xenograft tumor in MTD-DDP group were labeled by CD133 antibody and ALDH1 antibody in turned. CD133+ALDH1+ cells and CD133-ALDH1- cells were detected and sorted by fluorescence-activated cell sorting. These cells expressed strong fluorescence were CD133+ALDH1+ cells; these cells expressed weak fluorescence were CD133-ALDH1- cells.8 Observed the differences of VM fromation in 3DCC model by the CD133+ALDH1+ cells and CD133-ALDH1-.9 Statistical methods: Data were evaluated using SPSS17.0 statistical software, and data were expressed as mean ± standard deviation. The significance difference was determined using one-way ANOVA, S-N-K and Dunnett’s T3 method. α=0.05 is size of test. When P<0.05, the difference was statistically significant; when P > 0.05, there was no statistically significant difference.Results:1 The LDM chemotherapy schedule showed a more significant inhibition than MTD of the primary tumor cells to form VM in the 3DCC model.The MTD group of primary tumor cells formed the tube structures(VM) needed only about six hours, while the control group and LDM group were nine hours and twelve hours respectively. Because of the nutritional ingredient of 3DCC model was limited,the number of tube structures would stabilized after cultured 48 hours. Counting and comparing the vasculogenic mimicry density(VMD)could found that the difference between each groups, which had statistical significance(F=24.761;P =0.000).2 The MTD chemotherapy schedule showed a more significant proliferation ability than LDM of the primary tumor cells.Compared to LDM group and the control group, the MTT Cell growth curve of MTD group was higher since the third day(F=5.738; P =0.018), while the difference between LDM group and the control group had no statistical significance until the fifth day(P=0.853).3 The cells staining crossed the membrane of transwell migration testThe result of Transwell for invitation showed that the number of LDM group got through the maxtrix film compared with blank control group was significantly reduced, while the MTD group was statistically increased(F =47.570;P =0.000). This indicated that LDM model significantly inhibites the invasion ability of SKOV3 primary tumor cells.4 Expression of cancer stem cell marker CD133 and ALDH1The result of Western-blot suggest that the expression levels CD133 and ALDH1 proteins in primary tumor cells of MTD group were significantly higher than those in the control group and LDM group(F=8.251;P =0.006)(F=67.323;P =0.000), while the proteins expression levels in LDM group having no difference with control group(P=0.137)(P=0.517).5 The result of FCASThe primary cells of xenograft tumor in MTD-DDP group were cultured enough to sorted because of its CD133 and ALDH1 proteins expression levels were higher than others. The cells were double labeled, detected and sorted by fluorescence-activated cell sorting. Read the result from the FACS Diva software: the percentage of CD133+ALDH1+ was 1.82% and the percentage of CD133-ALDH1- was 79.25%.6 The CD133+ALDH1+ cells could formed VM with the low density in short time,while the CD133-ALDH1- cells needed higher density and more time.When the CD133+ALDH1+ cells and CD133-ALDH1- cells were cultured with the same density of 5×104/ml, the former could formed a great many of tube structures(VM) in 6 hours comparatively the latter didn’t form any though 48 hours had passed. There was a little of tube structures could observed after 24 hours when the CD133-ALDH1- cells were cultured with the density of 1×105/ml.Conclusion:1 The primary cells which accepted MTD chemotherapy showed higher abilities of proliferate, invasion and to form VM than LDM at the same total cumulative dose.2 The expression of tumor stem cell marker CD133 and ALDH1 in LDM group were significantly lower than MTD, it suggested that tumor cells with partial tumor stem cell properties to be enriched easier in MTD than the other two groups. These cells could influence the tumor blood supply and result bad prognosis.