• Sherman Medina posted an update 3 months, 4 weeks ago

    Specificity of the PCR product was confirmed by melting curves. Each experiment was carried out in triplicate. The data are presented as means ± standard this website errors (SE). The level of significance was set at a P < 0.05. Comparison of differences between treated and control groups in cell cycle and apoptosis assays was performed using student's t-test. All statistical tests were two-sided. Figure 2 shows that the IC50 of FKB for the uterine leiomyosarcoma cell line, SK-LMS-1, was approximately 1.25 µg/mL. At a dose of 5 µg/mL (8.8 µM), FKB inhibited the growth of SK-LMS-1 and ECC-1 cell lines by approximately 80%, but resulted in only a 10% growth reduction for the non-malignant uterine fibroblast cell line, T-HESC (Student’s t-test, P < 0.01). This result indicates a certain degree of specificity in FKB's ability to inhibit the growth of uterine LMS. To evaluate the mechanism for the cell growth inhibitory effect of FKB, the morphology of control and FKB treated cells was examined using light microscopy. As evidenced in Figure 3a, treated cells exhibited typical apoptotic morphologic changes including separation from surrounding cells, cell shrinkage and cell rounding.19 FACS analysis showed that FKB treatment at doses of 2.5 and 5 µg/mL (4.4 and 8.8 µM, respectively) resulted in an increase in both early (lower right, Annexin V-positive/PI-negative) and late (upper right, Annexin V-positive/PI-positive) apoptotic cell fractions as compared to control treated cells in SK-LMS-1 and ECC-1 cell lines (35.4 ± 3.1% and 29.1 ± 1.3% versus 0.02 ± 0.009 and 0.008 ± 0.004, respectively; P < 0.0001) (Fig. 3b). We also examined whether the growth inhibitory effect of FKB was associated with cell cycle arrest.14 As shown in Figure 4, FKB treatment resulted in a marked increase in the number of cells arrested at the G2/M phase in both SK-LMS-1 and ECC-1 cell lines [19.2 ± 0.34 to 35.6 ± 1.3% in SK-LMS-1 and 40.5 ± 1.1 to 54.9 ± 1.2% in ECC-1, P < 0.001 at an FKB dose of 2.5 µg/mL (4.4 µM)]. Apoptosis can be induced via the extrinsic pathway, through cell surface death receptor stimulation, or through the intrinsic pathway mediated by mitochondrial dysfunction.20,21Figure 5 illustrates that FKB treatment of SK-LMS-1 cells resulted in increased expression of death receptor (DR5) and the mitochondrial pro-apoptotic proteins Bim and Puma, while down-regulating the expression of an IAP, survivin. Analogously, FKB treatment of ECC-1 cells resulted in a significant increase in mRNA and protein expression for DR5 and a down regulation of survivin expression. Taken together, this data implies that FKB activates both DR5, and mitochondrial-mediated apoptotic pathways and decreases survivin expression, exhibiting a robust apoptotic mechanism against uterine LMS. Currently, adjuvant treatment of uterine leiomyosarcoma utilizes gemcitabine-docetaxel combination chemotherapy.