* < 0.05; *** < 0.001, as compared with untreated cells. 2.7. in the range of 10C25 M concentration and 25 M concentration for MIA PaCa2 according to MTT results. Interestingly, T1 complex was shown selective cytotoxicity even at concentration of 50 M. As T1, T2, and T3 showed equal potency ONX-0914 as cytotoxic agents on cancer cell lines (Table 2), T1 was selected to examine the mechanisms underlying pharmacological effects of this complex. Table 2 Cytotoxic activity of vanadium complexes on ONX-0914 PANC-1, MIA PaCa2 and hTERT-HPNE cell lines after 48 h of treatment. Data are expressed as IC50 and logIC50 (mean SD of 3 separate determinations) and were calculated on the basis of MTT and NR determinations. MTT Assay PANC-1 MIA PaCa2 hTERT-HPNE IC50 < 0.001, as compared with untreated cells. 2.6. Effects of T1 on Necrosis and Apoptosis The release of LDH from pancreatic cells was used to measure the effects of T1 on necrosis and late stage apoptosis [36]. Incubation of pancreatic cancer cells with T1 vanadium complex for 48 and 72 h did not release LDH (Figure 4) from PANC-1 cells. In contrast, T1 caused small, but a significant release of LDH from MIA PaCa2 and hTERT-HPNE cells (Figure 4). Open in a separate window Figure 4 LDH release from PANC-1, MIA PaCa2 ONX-0914 and hTERT-HPNE cells after 48 h and 72 h of incubation in the presence of the T1 complex. Data are mean SD of 3 separate determinations. * < 0.05; *** < 0.001, as compared with untreated cells. 2.7. Effects of T1 on ROS Generation Figure 5 shows that T1 induced ROS generation in pancreatic cells a concentration-dependent manner. Of note, increased generation of ROS in hTERT-HPNE cells was only detected at 50 M T1. Gemcitabine, which has been shown to decrease the viability of PANC-1 and MIA-PaCa2 cells through increased generation of ROS [37], used it as a positive control. Open in a separate window Figure 5 The levels of ROS induced by T1 vanadium complex in PANC-1, MIA PaCa2 and hTERT-HPNE cells following incubation for 48 h. Gemcitabine ONX-0914 was used as a positive control. Data are mean SD of 3 separate determinations. ** < 0.01; *** < 0.001, as compared with untreated cells. 2.8. Effects of T1 on Cell Cycle in Pancreatic Cells Flow cytometry and Western blot were used to measure the effects of T1 on cell cycle. Figure 6A shows that T1 resulted in G2/M cell cycle arrest in cancer cell lines. In contrast, the arrest in hTERT-HPNE cells was only observed at 50 M T1. Consistent with these findings, the expression of cyclinB1 and cdk1 proteins in cancer cells was significantly increased after treatment with T1 complex for 24 h and 48 h (Figure 6B). Open in a separate window Open in a separate window Figure 6 The cell cycle analysis of PANC-1, MIA PaCa2 and hTERT-HPNE cells treated with vanadium complex T1 after 24 h and 48 h of incubation. (A) The percentage of cells in each phase. (B) Western blot of cyclinB1 and cdk1 expression in cancer cells. Results are given as mean SD of 3 separate determinations. * < 0.05; ** < 0.01; *** < 0.001, as compared with untreated cells. 2.9. Effects of T1 on Autophagy and Binucleation in Cancer Cells Confocal laser scanning microscopy was used to evaluate morphology and autophagy in cancer cells treated with T1 (25 M). Untreated PANC-1 and MIA PaCa2 cells (Figure 7A) showed morphology typical of adherent cells. The structure of nuclei was disrupted in both cell lines after treatment with ONX-0914 T1 for 24 and 48 h. The observed binucleation (Figure 7A white arrows) is indicative of abnormal cell division and this could be a result of mitotic catastrophe [38]. No apoptotic bodies, which are characteristic for apoptosis, were observed [39]. Open in a separate window Figure 7 Morphological GREM1 features and the expression of LC3 protein following incubation of cancer cells with T1. (A) Immunocytochemical staining of PANC-1 and MIA PaCa2 cells exposed to T1 (25 M) for 24 and 48 h. (B) Western blot analysis of LC3 expression in PANC-1 and MIA PaCa2 cells exposed to T1 (1C50 M) for 24 h and 48 h. Gemcitabine was used as a positive.
