Effect Of Ursolic Acid On Proliferation Of T Cell Lymphoma Cell Lines And Its Mechanism

Objective:To investigate the effects and mechanism of ursolic acid for T cell lymphoma cell lines( Hut-78 and Jurkat)in vitro. Methods: 1. Inhibition of ursolic acid(10,20,40 or 80μmol/L) on Hut-78 and Jurkat cell proliferation was examined by MTT method. 2. Hut-78 and Jurkat cells were labeled with AnnexinⅤ/PI-FITC after ursolic acid(10,20,40 or 80μmol/L)effect, and apoptosis was analyzed by flow cytometry analysis. 3. After incubation with ursolic acid P65, p50, p52 and p100 proteins, and caspase-8, caspase-3 and caspase-9 protein expression in Hut-78 and Jurkat cells after drug treatment(10,20,40 or 80μmol/L ursolic acid)were detected by western blot methods. 4. After cultivated with ursolic acid( 10,20,40 or 80μmol/L), concentration of VEGF and COX-2 in Hut-78 and Jurkat cells supernatants was measured by ELISA methods. VEGF and COX-2 m RNA expression of Hut-78 and Jurkat cells after ursolic acid action were analyzed by reverse transcription polymerase chain reaction(RT-PCR). 5. After ursolic acid effect(10,20,40 or 80μmol/L), the supernatant of Hut-78 or Jurkat cells culture and umbilical vein endothelial cell line(HUVEC) were cultured together to detect the tubule number in Matrigel culture system. The result: 1. It was showed that ursolic acid inhibited the proliferation of Hut-78 and Jurkat cells(p<0.05), which related to the dose and duration of drug action(p<0.01). 2. Apoptosis of Hut-78 and Jurkat cells can be induced by ursolic acid treatment(p<0.05), which related to the dose and duration of drug action(p<0.01). 3. Expression of p65 and p50 protein(Hut-78 or Jurkat cells) were down-regulated by ursolic acid(10,20,40 or 80μmol/L)(p<0.05), in dosedependent manner(p<0.01), but not in p52 and p100. Moreover, ursolic acid up-regulated expression of caspase-8, caspase-3 and caspase-9 in dosedependent manner(p<0.05). 4. Concentration of VEGF and COX-2 in Hut-78 and Jurkat cells supernatants was down-regulated by ursolic acid(p<0.05) in dose-dependent manner(p<0.01). With RT-PCR methods, it was showed that VEGF and COX-2 m RNA expression of Hut-78 and Jurkat cells decreased by drug treatment(p<0.05) in dose-dependent manner(p<0.01). 5. After ursolic acid treatment, cultured with the conditional medium of Hut-78 and Jurkat cells,the number of tubules decreased(p<0.05) in Matrigel culture system in dose-dependent manner(p<0.01). In conclusion: 1. Proliferation inhibition of Hut-78 and Jurkat cells proliferation may be related to apoptosis induction by ursolic acid, which is complete with death receptors and mitochondrial pathways, and angiogenesis is also involved. 2. Ursolic acid mechanism may be accomplished by classical NF-κB pathway.