Table 1.
Cytotoxic activity of melphalan and topotecan on retinoblastoma cells after conventional and metronomic treatment.
Fig 1.
Effect of chemotherapy treatment schedule on retinoblastoma cell proliferation.
Growth inhibition assay performed on Y79, WERI-RB1, HSJD-RBT-7, and HSJD-RBT-8 cell models, respectively, using MTT after 72-h incubation of a conventional regimen (full triangle symbols) and metronomic treatment (open square symbols) for 7 days with different concentrations of topotecan (left) or melphalan (right). All symbols represent % of cell proliferation as compared to untreated control cells, expressed as means (SEM) of three independent experiments, each performed in triplicate.
Fig 2.
Effect of conventional and metronomic chemotherapy on proliferation of endothelial cells.
Growth inhibition assay performed on HUVEC and EPC after 72-h incubation of a conventional regimen (full triangle symbols) and metronomic treatment (open square symbols) for 7 days with different concentrations of topotecan (left) or melphalan (right). Symbols represent % of cell proliferation as compared to untreated control cells, expressed as means (SEM) of three independent experiments, each performed in triplicate.
Table 2.
Cytotoxic activity of melphalan and topotecan on endothelial cell types after conventional and metronomic treatment.
Fig 3.
In vitro angiogenesis assay using Matrigel to characterize the antiangiogenic effect mediated by treatment scheme with topotecan and melphalan.
Representative phase-contrast micrographs of tubular structures in cultured HUVEC (A) and EPC (B) previously exposed to conventional single-dose (upper row) or continuous treatment for 7 days (lower row) with topotecan at the IC25 (conventional IC25, 15nM; metronomic IC25, 0.5nM) or melphalan at the IC15 (conventional IC15, 5μM; metronomic IC15, 0.5μM), respectively. Magnification: 40X; Scale bar: 250μm. Experiments were performed in triplicate wells and five areas from each well were analyzed. The bar graph illustrates the significant decrease in the percentage of branch points after topotecan or melphalan used in a conventional or metronomic fashion compared to PBS-treated (control) cells or between treatment schedules. Data are shown as mean (SE) of three independent experiments; *p<0.05. Abbreviations: CC, control cells; CV, conventional treatment; MT, metronomic treatment.
Fig 4.
Effect of treatment schedule of chemotherapy on apoptosis of retinoblastoma and endothelial cells.
Retinoblastoma and endothelial cells were cultured at the conventional or metronomic IC50 of melphalan or topotecan. The rate of apoptosis was assessed by flow cytometry after double-staining with Annexin V-FITC and PI. (A) Representative dot plot of Y79 after conventional and metronomic treatment with topotecan or melphalan. Lower left and right quadrants represent viable (Annexin V-/PI-) and early apoptotic (Annexin V+/PI-) cells, respectively; the upper right and left quadrants show late apoptotic (Annexin V+/PI+) and necrotic (Annexin V-/PI+) cells, respectively. Numbers in each quadrant represent percentages of cells. (B, C) Percentages of viable (grey bars), early apoptotic (white bars), late apoptotic (dashed bars), and necrotic cells (black bars) are shown after conventional and metronomic treatment of Y79 and WERI-RB1 with (B) topotecan or (C) melphalan. Thereafter, we studied the impact of (D) topotecan or (E) melphalan conventional and metronomic treatment on HUVEC and EPC apoptosis. (F) Representative images of the morphologic changes of Y79 after conventional (upper row) or continuous treatment (lower row) with melphalan or topotecan observed by fluorescent microscopy (Magnification: 200X). Each experiment was performed in triplicate and repeated three times. Percentages correspond to mean (SD). Abbreviations: CC, control cells; CV, conventional treatment; MT, metronomic schedule.
Fig 5.
Effect of chemotherapy schedule on the expression of ABC transporters in retinoblastoma cell line Y79 and EPC cells.
Relative ABCB1 (left), ABCC1 (middle), and ABCG2 (right) expression level after exposing Y79 (A), EPC (B), respectively, to melphalan or topotecan conventional or metronomic treatments. All experiments were repeated at least three times. The 2−ΔΔCt method was used to analyze the relative change. Data represent mean ± SD of at least three experiments.
Fig 6.
Effect of treatment on retinoblastoma xenograft tumor growth.
Symbols show the tumor volume of (■) PBS-treated, (●) Maximum tolerated dose topotecan (3 mg/kg i.p once a week for two weeks), and (▲) Metronomic topotecan schedule of treatment (0.6 mg/kg i.p five days a week for two weeks). Symbols and bars represent mean ± sem. * p<0.05 with respect to maximum tolerated dose or PBS treated tumors.