| Objective:This study was initiated to evaluate the therapeutic effect of trichosanthin (TCS) on the growth of the A20 murine B-cell lymphoma in BALB/c-mice and to analyze its mechanism of action in cytotoxicity, immunoregulation and anti-angiogenesis, which could provide exact reference data for further research and exploration in TCS anti-lymphoma action and mechanism.MethodsInduced A20 murine B-cell lymphoma model in BALB/C mice, and then tumor-bearing mice were randomized to four groups (each group n=10) and treated with TCS 0.2,0.4, 0.8mg/kg/d dissolved in 0.2mL NS and administered by peritoneal injection for 7 days, as well as 0.2mL NS as control group. The volume of the tumors and the mice weight were measured. In addition, tumor-bearing mice’s survival time was observed.Starting from the cytotoxicity, immunity adjustment and anti-angiogenesis, this paper probes into the mechanism of TCS underlying anti-lymphoma activities. In order to observe the cytotoxic activity of TCS to A20 cell lines in vitro, A20 were cultured with different concentrations of TCS (2.5,5,10,20,40μg/mL) for 24-72h, and the inhibitory rate of cell proliferation was detected by CCK-8 method. The cell cycle and apoptosis of cells treated with various concentrations was determined by flow cytometry (FCM). In vivo experiments as described above, the serum levels of TNF-a, IL-2 and IFN-γ were tested by ELISA. The percentage of T-cells, B-cells and NK cells were quantified in the spleen of individual mice by FCM.To study the effect of TCS’s anti-tumor angiogenesis, the microvessel density (MVD) in tumor tissue was calculated by HE staining and immunohistochemical staining and morphometric technique. The expression of vascular endothelial growth factor (VEGF) was assayed by immunohistochemical staining. The serum levels of matrix metalloproteinases (MMP)-2 and MMP-9 of tumor-bearing mice were tested by ELISA.The concrete anti-angiogenic mechanism of TCS were studied. The mRNA and proteins of VEGF and bFGF expressions was detected by using reverse transcription-polymerase chain reaction(RT-PCR) and western blot respectively. HUVECs were cultured in vitro with different concentrations of TCS for 24-72h, and the inhibitory rate of proliferation was detected by CCK-8 method. The cell cycle and apoptosis of cells was determined by FCM. Using scratch test and Matrigel coated Transwell transmembrane compartment model to assess the migration and the invasive ability of HUVECs at different concentrations of TCS (0,1.5,3,6μg/mL). Finally, chick chorioallantoic membrane (CAM) assay was used to verify the activity of TCS’s anti-angiogenesis. Result1. TCS inhibit mouse A20 lymphoma growth in vivoThe tumor volume in the high (0.8mg/kg) and middle (0.4mg/kg) dose group was 367±31.6mm3 and 516±39.3mm3,and much smaller than those in the control (1215±98.91mm3) and the low dose TCS(0.2mg/kg) group (1005±81.56mm3) (P＜0.05). The tumor weight in the high and middle dose group was 1.55±0.17g and 0.968±0.26g, and lighter than those in the control (2.43±0.62g) or low dose group (0.366±0.08g) (P＜0.05). The growth inhibitory rate in three experimental groups were 21.2%,46.2% and 60.1%, respectively. The median survival time in the high and middle dosage group was 42±5 and 34±3d days, respectively, and was longer than it in the control(24±2d) and the low dose group (25±2 d) (P＜0.05).2. TCS showed no direct cytotoxic effect on A20 in vitro and no effect on the immunological function bof the tumor-bearing miceStudies showed that TCS had no inhibitory effect on A20 cells and no effect on cell cycle distribution or apoptosis in vitro experiments (P>0.05). The animal experimental res ults showed that TCS had little impact on T lymphocyte subgroup, B lymphocyte or NK cells ratio in spleen of all mice (P>0.05).There was no significant difference in serum levels of tumor immunity-related cytokines among among groups, such as TNF-a, IL-2 an d IFN-γ (P>0.05)3. TCS showed strong inhibition of angiogenesis in mouse lymphoma models and in CAMThe MVD based on HE staining in the control and three experimental groups were 31±7, 24±5,12±4 and 9±3 Vessels/HPF, respectively. Following immunofluorescence staining tests, the MVD in the four groups were 38±4,32±3,13±3 and 10±2 Vessels/HPF, respectively. A statistical analysis was performed on these data indicated that middle and high dosage of TCS could obviously reduce the MVD at a statistical significant extend when compared with the two other groups (P<0.05 or P＜0.01)The serum level of MMP-2 and MMP-9 in control mice was 83.56±16.35ng/mL, and 27.76±7.8ng/mL, respectively, and 67.58±11.04ng/mL and 21.84±7.23ng/mL, respectively in low dose of TCS group. Middle and high dosage of TCS (MMP-2:41.28±7.04ng/mL, 27.22±4.38ng/mL, respectively) (MMP-9:14.02±3.39ng/mL,7.58±2.08ng/mL, respectively) could obviously reduce the level MMP-2 and MMP-9 at a statistical significant extend when compared with the other two groups (P<0.05). RT-PCR and Western blot results showed that VEGF mRNA and protein, bFGF mRNA and protein in A20 were significantly lower in TCS groups than control group with significant difference (P<0.05 or P<0.01). TCS (0.5μg/egg, 1.0μg/egg,2.0μg/egg) inhibited angiogenesis in CAM by (72±18.1)%, (57±10.3)% and (45±6.1)%, more efficiently than it in control group (P＜0.05 or P＜0.01).4. Inhibition of TCS on the biologic activity of HUVECsTCS significantly inhibited the growth of HUVECs in vitro and the inhibition was dependent on the drug concentration and action time. The IC50 of HUVECs by TCS for 24,48 and 72h was 27.39±0.8μg/mL,14.04±0.03μg/mL and 5.9±0.2μg/mL, respectively. The early and late apoptosis rate of HUVECs treated with different concentration of TCS (5,10,15μg/mL) for 48h was (17.1±2.6)%, (32.6±3.6)% and (52.1±5.3)%, respectively, and the difference was very remarkable (P＜0.01) compared with the control group. FCM results showed that the ratio of cells in G0/G1 phase was (73.5±4.89)%, (74.2±4.2)% and (76.8±4.32)%, respectively, which was higher than that in the control group (P<0.05).Compared with the control group, the wound diameter in HUVECs treated with TCS (1.5,3, 6μg/mL) for 24h increased by (26.5±5.9)%, (35.7±5.1)% and (66.2±7.3)%, respectively, and increased by (21.5±9.5)%, (68.1±7.3)% and (173.5±20.5)% for 48h. TCS in above dosage scope reduced the invasion capacity of HUVECs by (63.65±2.53)%, (56.18±2.62)% and (42.43±3.04)%(P＜0.05, P＜0.01)..ConclusionTCS could inhibit significantly the A20 murine B-cell lymphoma growth and prolonged the survival period of tumor-bearing mice. Preliminary study of the mechanisms of action of TCS demonstrated it could inhibit significantly angiogenesis, but it had no effect on the proliferation of A20 cells and on the immune function of tumor-bearing mice. Initial studies showed that the pathway of involved in its anti-angiogenesis mainly included as follow. Firstly, TCS inhibited the expression and the secretion of VEGF and bFGF from A20 lymphoma cells. Secondly, TCS decreased the level of MMP-2 and MMP-9 in the extracellular matrix microenvironment. Thirdly, TCS inhibited HUVECs proliferation, invasion and migration, and induced apoptosis of HUVECs.SignificanceThis research has firstly affirmed the anti-lymphoma value of TCS by in vivo experiment study, and then investigated the mechanism of TCS’s anti-lymphoma actions from the perspectives of cytotoxicity, immunity adjustment and anti-angiogenesis. The impact of TCS on proangiogenic factors released by lymphoma cells, extracellular matrix metalloproteinase and endothelial cells were observed at the cellular and molecular levels and the mechanis m of its anti-angiogenesis was defined preliminarily. The present study provided experiment al and theoretical basis from an objective point of view on "TCS is an effective metho d in the treatment of lymphoma"... |
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