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Abstract
Objective
The aim of this study was to detect the levels of M30-antigens as a biomarker of apoptosis in cells and their culture media after treatments with anticancer drugs as a preclinical study.
Methods
After HeLa and OVCAR-3 cells were treated respectively with paclitaxel, cisplatin, and camptothecin, the harvested cells were stained sequentially with M30 monoclonal antibodies and propidium iodide (PI). Afterwards, they were analyzed using a FACScan flow cytometer and observed under an immunofluorescence microscope for M30-FITC immunofluorescences. Levels of M30 antigens were also detected in their culture media using M30-Apoptosense ELISA kit.
Results
The levels of M30-FITC immunofluorescences were elevated in both cell lines after each drug treatments compared with those of control cells. The levels of M30 antigens detected by ELISA in media culturing each cell line treated with each of drugs were elevated compared with those of control cells.
Figures and Tables
Fig. 1
DNA histograms (left) and linear diagrams (right) showing the changes of cell cycle phases and apoptotic populations (sub-G1 peaks) stained with propodium iodide (P1) and measured by FACScan flow cytometer in HeLa and OVCAR-3 cells. The blue peaks represent apoptotic populations (sub-G0G1 peaks). The first red peaks). The first red peaks represent the G0G1 phases of the cell cycles. The second red peaks represent G2M phases of cell cycles. The mid portions between two red peaks with diagonal lines represents S phases of cell cycles. (A) Untreated control cells, (B) cells treated with 1,000 nM of paclitaxel, (C) cells treated with 250 ng/mL of cisplatin, and (D) cells treated with 30 nM camptothecin. (E) and (F) The linear diagrams showing distributions of cell cycle phases and apoptotic sub-G0G1 peaks in HeLa and OVCAR-3 cells teated with 1,000 nM of paclitaxel, 250 ng/mL of cisplatin, and 30 nM of camptothecin.
Fig. 2
The histograms are showing the levels of M30-FITC immunofluorescences (Number vs M30-FITC immunofluorescence) detected by flow cytometer in HeLa and OVCAR-3 cells treated with anti-cancer agents and the comparisons between sub-G0G1 phase fractions and positive M30-FITC immunofluorescences. The popilations in gate R2 indicate negative M30-FITC immunofluorescences and those in gate R3 indicate the apoptotic populations with positive M30-FITC immunofluorescences. (A) Untreated control cells, (B) cells treated with 1,000 nM of paclitaxel, (C) cells treated with 250 ng/mL of cisplatin, and (D) cells treated with 30 nM of camptothecin. (E) and (F) The correlation between the detected levels of sub-G0G1 fractions and M30-FITC immunofluorescences were evaluated by Spearman's Methods (correlation coefficient=0.59848, P=0.0003). There were no significant differences between them when compared by Wilcoxon Rank Sum Test.
Fig. 3
The M30-FITC immunofluorescences observed under the immunofluorescence microscopy. (A) Untreated control HeLa and OVCAR-3 cells. There can be seen some positive cells with scanty intracytoplasmic M30-FITC immunofluorescences compared to the treated cells. (B~D) Cells treated with 1,000 nM of paclitaxel, 250 ug/mL of cisplatin, and 30 uM of camptothecin in order. There can be seen many cells with strong intracytoplasmic immunofluorescences in all of the treated cells, PAC: paclitaxel, CIS: cisplatin, CAM: camptothecin.
Fig. 4
The levels of M30 antigen in each cell line groups.
This figure showing the comparison between the levels of CK18-Asp396 (M30) antigen detected by ELISA in the culture media of untreated controls and HeLa and OVCAR-3 cell lines treated with 1,000 nM of paclitaxel, 250 ng/mL of cisplatin, and 30 uM of camptothecin. PAC: paclitaxel, CIS: cisplatin, CAM: camptothecin.
Table 1
The percentages of cell cycle phases and the detected levels of sub-G0G1 phase fractions and positive M30-FITC immunofluorescences representing apoptosis in HeLa and OVCAR-3 cell lines after treatments with paclitaxel, cisplatin and camptothecin, respectively.
The HeLa and OVCAR-3 cells were treated with 1,000 nM of paclitaxel, 250 ug/mL of cisplatin, and 30 uM of camptothecin, respectively. The decreases or increases of fractions of G0G1, S and G2M phases of cell cycles from control were compared by the Wilcoxon Rank Sum Test (*P<0.05). The decreases or increases of fractions of sub-G0G1 and M30-FITC reflecting apoptosis were also compared from control by the Wilcoxon Rank Sum Test (*P<0.05). There were positive correlations between the values of sub-G0G1 fractions of cell cycles and M30-FITC immunofluorescences when they were analyzed by the Spearman's Correlation (correlation coefficient=0.59848, P=0.0003). There were no significant differences between the levels of sub-G0G1 fractions of cell cycles and M30-FITC immunofluorescences representing apoptosis observed after treatments, when they were compared by Wilcoxon Rank Sum Test.
Table 2
Levels of apoptosis detected by ELISA using M30 (CK18-Asp396) antibody in the culture media after treatments with 1,000 mM of paclitaxel, 250 ug/mL of cisplatin, and 30 uM camptothecin in HeLa and OVCAR-3 cell lines.
The differences among anti-cancer agents including controls were significant statistically when were evaluated by 2-Way ANOVA Test (*P<0.0001). The detected levels of M30-antigens were not different significantly between HeLa and OVCAR-3 two cell lines. The levels of M30-antigens detected after treatments with three agents were different from those of controls when were evaluated by unpaired T-Test (†P<0.05). The detected levels of M30-antigens were much higher in both of cell lines treated with paclitaxel and cisplatin than in those treated with camptothecin when compared to controls, when were evaluated by Schffe's Test. But there were no significant differences between paclitaxel and cisplatin.
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