Statistical analysis
Data were presented as mean±SD using SPSS 11.0 statistic software, Paired t-test was used to compare two means from one group and q-test was used for comparison of multiple groups. P value less than 0.05 was considered as statistically significant.
Results
Effects of triptolide on the proliferation of Raji cells by MTT
As shown in Figure 1, Treatment of Raji cells with different concentrations of triptolide for 12, 24, 36, 48, 60 and 72 hours, resulted in significantly enhanced cytotoxicity in dose- and time-dependent manner. After treated with concentration of (3.13, 6.25,12.50, 25.00, 50.00) nmol/L triptolide for 24 hours, inhibitory rates were 127%, 15.6%, 31.5% and 52.8%, 53.3% and 60.3% respectively. IC50 value was 25 nmol/L.
Quantification of VEGF production by ELISA
Raji cells released VEGF protein constitutively. As shown in Figure 2. VEGF production was significantly increased by TNF-α, as compared with control (P<001). Triptolide reduced the VEGF production compared with control (P<0.01).
VEGF mRNA expression in Raji cells
As shown in Figure 3, after incubation with different concentrations of triptolide (12.5, 25.00, 50.00 nmol/L) for 24 hours, VEGF165 and VEGF121 mRNA could be detected, and the mRNA expression ratios (VEGF121/β-actin) were 0.48±0.08, 0.24±0.01 and 0.05±0.000, respectively, while the mRNA expression ratio (VEGF121 /β-actin) in control group was 0.67±0015. The mRNA expression ratios (VEGF165/ β-actin) were 0.57±0.01,0.30±0.02 and 0.08±0.01, respectively, while the mRNA expression ratio (VEGF165 /β-actin) in control group was 0.71±0.01. We also tested VEGF mRNA in Raji cells treated by TNF-α (10 ng/ml) , the result showed that compared with the control, TNF-α augmented the expression of VEGF165 mRNA (P＝0.00, P<0.01) and VEGF121 mRNA (P＝0.007, P<0.01)， but triptolide reduced the expression of VEGF165 mRNA (P＝0.003, P<001) and VEGF121 mRNA (P＝0.008, P<001) (n=3).
Matrigel network formation assay
After ECV304 cells were seeded on the Matrigel, network formation was initiated in about 24 hours. No network and cord formations were seen in control and triptolide group. There was tube formation on Matrigel in the supernatant of Raji culture group and the supernatants of groups treated by VEGF and TNF-α in Raji cells. No network and cord formations were seen when the 20 ng/ml TNF-α directly added to ECV304 culture (Figure 4).
Discussion
Triptolide, a traditional Chinese medicine, has been reported to be effective in the treatment of auto-immune diseases, and it can also induce anti-neoplastic activity on several human tumour cell lines. This study investigated the cytotoxic function and the functional mechanism of triptolide on Raji cells. The results of study showed that triptolide induced antiproliferative effects on Raji cells, Raji cells released VEGF protein constitutively, VEGF production was significantly increased after treated by TNF-α, compared with control, triptolide reduced the expression of VEGF mRNA in dose-dependent manner, compared with control, and TNF-α augmented the expression of VEGF mRNA. Some reports showed that triptolide, a purified compound from Tripterygium, possessed antitumour properties and induced apoptosis in a variety of malignant cell lines. Chan ［7］ found that triptolide induced cytotoxic effects on human promyelocytic leukemia, T cell lymphoma and human hepatocellular carcinoma cell lines. TNF-α can up-regulate VEGF expression at the mRNA and protein levels. Koyama ［8］ reported that BEAS-2B cells and A549 cells released VEGF constitutively. Interleukin IL-1b and tumour necrosis factor TNF-α augmented the release of VEGF in a time- and dose-dependent manner. The released VEGF was VEGF165. Some reports showed that triptolide can inhibit VEGF expression and production ［5,9］. Our results were coincident with these reports. After ECV304 cells were seeded on the Matrigel, no network and cord formed in control and triptolide group. There was tube formation on Matrigel in the supernatants of Raji culture group and the Raji culture supernatants treated by VEGF and TNF-α . VEGF can caused endothelial tube formation in HUVECs［10,11］. TNF-α also can caused endothelial tube formation in HUVECs［12］. Our results were coincident with some reports, but there was no tube formation on Matrigel in the Raji cultur supernatants treated by triptolide. Vasculogenesis and angiogenesis are processes of the formation of new vascular networks. VEGF plays a crucial role in angiogenesis. VEGF family members and their receptors seem to have important action during early vasculogenesis and angiogenesis. In our study, only the mRNA expression of VEGF165 and VEGF121 were detected in Raji cells, and We know that the gene for human VEGF is organized into eight exons. As a result of alternative splicing, at least four transcripts encoding mature VEGF have been detected, encoding VEGF containing 121, 165, 189, and 206 amino acid residues (VEGF121, VEGF165, VEGF189, and VEGF206), and a 26 amino acid signal peptide preceeded in each. VEGF121 and VEGF165 are diffusible proteins that are secreted into medium. VEGF189 and VEGF206 have a high affinity for heparin and are mostly bound to heparin-containing proteoglycans in the extracellular matrix. Transfection studies demonstrate that VEGF121 and VEGF165 are secreted and exhibit both mitogenic and peameability-inducing activities, but VEGF189 and VEGF206 remain primarily cell associated and lack the mitogenic activity of the smaller form, suggesting that VEGF165 and VEGF121 are pivotal forms of VEGF［13,14］. Our results were coincident with these reports.Above all, the expressions of VEGF in Raji cells were increased by TNF-α and suppressed by triptolide. VEGF and TNF-α can induce angiogenesis, and triptolide can inhibit angiogenesis in ECV304 cells. This study provides the first evidence for regulation of VEGF gene expression by triptolide and TNF-α and their effects on angiogenesis.
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