Figure 1.
Inhibition of proliferation of Jurkat, Hut78 and EL4 cells by 13-MTD treatment.
(A) Cultivation with 13-MTD for 48 hours at different concentrations (10, 20, 40, 60, 80 µg/ml) inhibited the proliferation of Jurkat, Hut78 and EL4 cells in a dose-dependent manner. (B) Cell viability of 13-MTD-treated Jurkat cells decreased in a time-dependent manner at different incubation time points (24, 48, 72 h).
Figure 2.
The effects of 13-MTD in the G1 phase arrest in T-NHL cells.
Cells at a density of 2 × 106 cells per well were treated with 13-MTD at different concentrations (0, 20, 40, 60, 80 µg/ml) for 48 h and harvested to evaluate the cell cycle distribution. The percentage of cells in G1, S, and G2 phases in Jurkat (A), EL4 (B) and Hut78 (C) cells are shown. *P>0.05, ** P<0.05 compared with the solvent group. All data are derived from three individual experiments with triplicate wells.
Figure 3.
13-MTD induces apoptosis, pro-caspase-3 and PARP cleavage in T-NHL cells.
Apoptotic cells were detected by flow cytometry after Annexin V/PI staining, and caspase-3 and PARP were detected by Western blot analysis. (NC, negative control) (A) 13-MTD induced apoptosis in Jurkat, Hut78, and EL4 cells after 48 h of 13-MTD treatment at different concentrations (0, 20, 40, 60, 80 µg/ml). The number of apoptotic cells increased in a dose-dependent manner. Values represent the means ± standard deviation (SD) of three independent experiments. (B) 13-MTD induced apoptosis in Jurkat cells at different incubation time points (24, 48, 72 h). Values represent the means ± SD of three independent experiments. (C) Jurkat, EL4 and Hut78 cells were incubated with 13-MTD or solvent control at 60 µg/ml for the indicated time periods. Each sample was stained with Annexin V/PI and analyzed by flow cytometry. The ratio of cells is shown in each quadrant. The percentage written behind the braces refers to the ratio of apoptosis cells in each sample. Data shown are representative of three independent experiments. (D) Jurkat,EL4 and Hut78 cells were treated with 60 µg/ml 13-MTD or solvent control for 2, 6, 12 and 24 h. Cells were then collected, lysed and subjected to western blot analysis with PARP and caspase-3 antibodies that can detect cleaved PARP and cleaved caspase-3. GAPDH was used as a loading control. All data are derived from three individual experiments with triplicate wells.
Table 1.
13-MTD induced apoptosis in Jurkat, Hut78 and EL4 cells.
Figure 4.
Western blot analysis of the expression of Bcl-2, c-myc, AKT, p-Akt, p-NF-κB in Jurkat EL4 and Hut78 cells treated with 60 µg/ml solvent control or 13-MTD for 2, 6, 12, 24 h.
GAPDH was used as a loading control. There was no change in the expression of Bcl-2, c-myc or AKT proteins. The expression of phosphorylated AKT and NF-κB were decreased in a time-dependent manner with 13-MTD treatment. All data are derived from three individual experiments with triplicate wells. (B) These three cell lines were treated with solvent or 13-MTD (60 mg/ml) for 24 hours, followed by Akt inhibitor V (40 uM) exposure for 30 min. The phosphorylation of AKT was significantly inhibited by Akt inhibitor V. The anti-growth (C) and apoptosis-promoting (D) effects of 13-MTD on T-NHL cells almost disappeared once the phosphorylation of AKT was inhibited. Result are representative of there independent experiments. All the values represent means ±standard deviation (S.D.).
Figure 5.
The therapeutic effect of 13-MTD on Jurkat cell xenografts.
The tumor volumes of xenografts were measured with calipers every 3 days for a total of 30 days after the start of treatment. After 30 days of treatment, mice were sacrificed and the tumors removed and photographed. (A) Tumor growth was significantly suppressed with 13-MTD treatment. The respective tumor volumes from the solvent control and 13-MTD treatment groups were 2325.43±318.32 mm3 and 1000.54±156.78 mm3 (n = 5, P<0.01, Student’s t-test) (B) Tumors were then fixed and stained with H&E to examine tumor cell morphology. IHC showed decreased phosphorylation of AKT and NF-κB in the tumor tissue after treatment with 13-MTD. (Original magnification×100) (C) 13-MTD enhanced the activation of caspase-3 and PARP proteins in tumor xenografts compared with controls. Tumor lysates were subjected to the analysis of protein levels using western blot analysis. GAPDH was used as a loading control. Representative blots are shown from independent experiments with six different tumors in each treatment group.