1) demonstrated that treatment with 1 μM and 2 μM for 48 hours insignificantly triggered cell death (P > 0.05 VS control). However, concentrations from 5 μM to 30 μM could markedly inhibit tumor cells (P < 0.01 VS control). The bivariate correlation analysis confirmed the negative relationship between PTL concentrations and cell survival rates and the positive relationship between PTL concentrations and cell inhibition rates. In BxPC-3 cells, EC50 was estimated to be 14.5 μM. Figure 1 PTL inhibited BxPC-3 proliferation. MTT assay demonstrated that PTL can inhibit BxPC-3 cell growth in vitro. Besides, this effect was in a dose-dependent manner. The cell viability and inhibition rates were calculated by comparing with
the control group (100%) Selleck BB-94 Selleckchem Necrostatin-1 after 48 hours treatment. Data were presented as mean ± SD (n = 3). Points, mean; bars, + SD. *, P > 0.05; **, P < 0.01 compared with the control group. PTL induced significant apoptosis in human pancreatic cancer cell To investigate the effect of inducting apoptosis by PTL in BxPC-3 cells, the flow cytometry and DNA fragmentation analysis were preformed. Annexin-V/PI-FACS analysis (Fig. 2A) was applied to quantify the apoptotic phenotype. Annexin-V-positive cells (right quadrant in the density dot plot) were summarized, including early apoptotic and late apoptotic cell death. PTL-treated cells revealed morphologic events of apoptosis more significantly than cells treated with DMSO alone. The inductive
effect of apoptosis presented as a concentration-dependent manner. The apoptosis induced was further confirmed using DNA fragmentation analysis (Fig. 2B). Disintegrated nuclei and nonrandom DNA fragmentation were found on gels. More apoptotic internucleosomal DNA fragmentation was observed after higher concentrations of PTL treatment. These results revealed that PTL effectively induced a dose-dependent apoptosis in human pancreatic cancer cell. Figure 2 PTL induced BxPC-3 apoptosis. BxPC-3 cells were
treated with the indicated concentrations of PTL for 48 hours. (A) The quantification of apoptosis was estimated by Annexin-V/PI-FACS analysis. As apoptotic events Annexin-V-positive cells (right quadrant in the density dot plot) were summarized. (B) DNA Fragmentation Thiamet G Analysis indicated that the cells treated with higher concentrations of PTL showed higher proportions of apoptotic internucleosomal DNA fragmentation. These results revealed that PTL-induced apoptosis in BxPC-3 cells was in a dose-dependent manner. The data was described as mean ± SD (n = 3) and the representative figures are shown. PTL suppressed BxPC-3 cell migration Increased migration rate is one of the characteristics in metastatic cancer cells [13]. Pancreatic cancer is a major health problem due to its high risk of metastasis. Accordingly the wound closure assay (Fig. 3) was used to investigate if PTL influenced migration ability of BxPC-3 cells. Wound gap of similar size was created in monolayer BxPC-3 cells at 0 hour.