| Multiple myeloma(MM) is a malignancy of the plasma cell characterized by migration and localization to the bone marrow where cells then disseminate and facilitate the formation of bone lesions. While conventional chemotherapy and novel agents, like IMiDs and proteasome inhibitor, is effective in palliating the disease, and even prolonging survival, this disease is generally regarded as incurable. 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 thatTPL possessed potential anti-tumor properties, and induced apoptosis of MM cell lines 8226 and U226 in our former research. In the present study we investigated the therapeutic effects and mechanisms of TPL against human MM Dex-resistant cell line MM.IR and Dex-sensitive cell line MM.1S.First of all, we used MTT assay to examine the effects of different concentrations of TPL on the growth of MM.IR cells, using its Dex-sensitive cell line MM.1S as control. 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 5.056ng/ml, 2.468ng/ml, 1.935ng/ml (MM.1S), and 9.879ng/ml, 4.914ng/ml, 3.635ng/ml (MM.IR) after the treatment of triptolide for 24, 48, 72 hours, respectively.Then, in order to investigate whether apoptosis is associated with the antitumor activity of TPL in human MM.l cells, we observed the cell morphology using Wright-6iemsa staining, evaluated the exposure of phosphatidylserine (PS) on MM cells after double staining with f luorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI), and analyzed the cell cycle using propidium iodide staining. We foundtypical apoptotic morphology such as pyknosis and apoptotic body after MM.l cells were exposed to TPL for 24hr. TPL induced a progressive increase in sub-GO/Gl 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 MM.l cells' apoptosis, we used western-blot to assay the protein expression of Bcl-2 family members. Bcl-2 and Mcl-1 protein level were down-regulated and Box protein level was up-regulated when 10-20ng/ml TPL was treated. The results indicated that TPL induced-apoptosis is mediated, at least in part, through changing the ratio of anti- and proapoptotic Bcl-2 family proteins.We further examined whether TPL enhances the growth inhibitory effect of Dex and PS-341. MM.1S and MM.1R cells were cultured for 24 hours with bex (0.5 u g/ml) or PS-341 (0.001-0.01 X 10'6M) in media with or without sub-ICso concentrations of TPL (1.25-5ng/ml). TPL enhances growth inhibition mediated by Dex and PS-341, as analyzed by MTT assay.In conclusion, TPL can inhibit the viability of human MM bex-sensitive cell line (MM.1S), as well as Dex-resistant cell line (MM.1R) in a dose-dependent manner. And this effect is induced by apoptosis. TPLtriggers the apoptosis of MM.l cell line, at least in part, through the mitochondrial pathway. TPL also overcomes the conventional drug resistant of MM cells, and enhances cytotoxicity of proteasome inhibitor PS-341.
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