The Effects Of Triptolide And Triptolide Combined With PS-341 On Hodgkin's Disease Cells

Hodgkin's Disease (HD) is one of hematologic malignant tumor ,which is source from lymph tissue and absorbent gland ,and it is related to the canceration of the cells that come from the cell differentiation and cell proliferationof leukomonocyte in the cource of immune response.The patho-characteristic of HD is that Hodgkin's cell and the Reed-Sternberg cell (RS) which appear in the backgrand of reactivity cell that include leukomonocyte,plasmocyte,histiocyte ,fibrocyte.The main clinic appearance is the lymphadenectasis without pain, appearing in cervical part first, and it can offend other absorbent gland with the development of this disease. Some patients also can show itch of skin. Although Hodgkin's Disease is thought that it can be cured, there are some patients who cannot be cured and some patients who can relapse. So other drugs should be found to be used to treat these patients. Triptolide (TPL) is a purified component extracted from Tripterygium wilfordii Hook. F (TWHF) and has been demonstrated to be effective in patients with a variety of inflammatory and autoimmune diseases, such as rheumatoid arthritis, nephritis and lupus erythematosus. Recent studies showed that TPL possessed potential anti-tumor properties, and induced apoptosis of MM cell in our former research.As we know,with lots of new chemotherapeutics drugs coming out,the proteasomes inhibitor Bortezomib (Bortezomib,PS-341, VELCADE) has been extensively used to treat the patients ,and it mainly decreases the level of NF k B or regulates cyclin and the signal apoptosis of tumor cell. In the present study we investigated the therapeutic effects and mechanisms of TPL and combining TPL with PS-341 against human Hodgkin's Disease cell line L428. First of all, we used MTT assay to examine the effects of TPL and TPL combining with PS-341 on the growth of L428 cells, The results indicated that cell viability in the presence of TPL decreased in a dose-dependent manner. The inhibitory rates of cell growth were positively correlated with TPL concentrations (P< 0.01). The growth-inhibitory IC₅₀ values were 901.31ng/ml; 12.13ng/ml; 8.686ng/ml after the treatment of triptolide for 24, 48, 72 hours, respectively.Then, in order to investigate whether apoptosis is associated with theantitumor activity of TPL and combining TPL with PS-341 in L428, we observed the cell morphology using Wright-Giemsa staining, evaluated the exposure of phosphatidylserine (PS) on L428 cells after double staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI), and analyzed the cell cycle using propidium iodide staining. We found typical apoptotic morphology such as pyknosis and apoptotic body afterL428 cells were exposed to TPL for 48h. TPL induced a progressive Increase in sub-GO/G1 phase cells in a dose-dependent manner. Apoptosis induced by TPL was also confirmed using Annexin V and PI staining to detect externalization of PS on the cell membrane.To further investigate the mechanism possibly involved in TPL-induced L428 apoptosis, we used western-blot to assay the protein expression of the caspase single and Bcl-2 family members. The raw tape of PARP, caspase-3, 9, Bcl-2 were down-regulated and the cleaved tape of PARP, caspase-3, 9, Bax protein level was up-regulated when 5-200ng/ml TPL was treated for 48h. The results indicated that TPL induced-apoptosis is mediated, at least in part through the exogenous parthway and changing the ratio of anti- and proapoptotic Bcl-2 family proteins.We further examined whether PS-341 could enhance the growth inhibitory effect of TPL. L428 cells were treated for24 hours by different concentrations of TPL(5-500 ng/ml) combined with 80 ng/ml PS-341. The growth-inhibitory IC₅₀ was 7.2462ng/ml.We could find thatPS-341 inhanced the effects of TPL as analyzed by MTT assay.Also the double staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI), analyzing the cell cycle using propidium iodide staining and the western-blot to assay the protein expression of the caspase single and Bcl-2 family members could show that PS-341 inhance the effects of TPL.In conclusion, TPL can inhibit the viability of L428 in a dose-dependent manner. And this effect is induced by apoptosis. TPL triggers the apoptosis of L428 cell line, at least in part through the mitochondrial pathway. PS-341 enhances cytotoxicity of TPL.