Effects And Mechanisms Of Triptolide And Homoharringtonine In Human Multiple Myeloma Cells

Human multiple myeloma (MM) is a presently incurable hematological malignancy, despite the use of conventional or high-dose chemotherapy with stem cell support. Incomplete response rates and the eventual emergence of drug resistance have been major obstacles to treatment with conventional therapies. Although the recent development of new treatment strategies based upon molecular targeting, such as proteasome inhibitors and thalidomide, have made great progress in the treatment of MM, new treatments are also urgently needed. We try to study the effects and mechanisms of triptolide and homoharringtonine in human multiple myeloma cells. There are two sections in our research.Section 1: Triptolide induces apoptosis in multiple myeloma cells involvement of cell signaling pathwayTriptolide (Diterpenoid triepoxide), a purified component of a traditional Chinese medicine, is extracted from a shrub-like vine named Tripterygium wilfordii Hook F (TWHF), and has been reported to be effective in the treatment of auto-immune diseases, such as rheumatoid arthritis, nephritis and systemic lupus erythematosus. It can also induce anti-neoplastic activity on several human tumor cell lines. There was no report of the anti-tumor potential and mechanism of TPL in multiple myeloma (MM) cells.Using MTT assay we first investigated the effect of TPL on RPMI8226 and U266 growth. Theresults showed that following treatment with TPL (10ng/ml) for 48 h, the proliferation of RPMI8226 and U266 cells were inhibited significantly, in comparison with the blank control group (P<0.05). 50% growth inhibition (IC50) at 48h in U266 and RPMI8226 occurred at about 25ng/ml. In addition, exogenous IL-6 could not overcome the growth inhibition induced by TPL in RPMI8226 cells. TPL was not synergetic or additive with dexamethasone in the growth inhibition of MM cells. But TPL enhanced the inhibitory effect of DOX on MM cell growth. More importantly TPL also inhibited the growth of freshly isolated MM cells from 3 patients with an IC50 (at 48h) of 20-40 ng/ml. In contrast, the IC50 of TPL was higher (>100ng/ml) in normal cells. To explore the possible mechanism of the inhibition of TPL on MM cells, further investigation was carried out from the two stratifications of inducing apoptosis and cell signaling pathway.In order to exactly analyze the cell apoptosis quantitatively and qualitatively, in addition to some classic and specific and sensitive methods, the flow cytometry was also used to evaluate the DNA content, percentage of hypodiploid and translocation of phosphatidyl serine (PS). It showed that RPMI8226 and U266 cells following treatment with 20ng/ml, 40ng/ml TPL for 48h, a typical DNA ladder was observed.; treatment with 20ng/ml, TPL for 48h, a progressive increase in sub-GO /G1 phase cells; and apoptosis induced by TPL was also confirmed by following annexinV-PI staining, RPMI8226 exposure to 10- 40 ng/ml TPL for 24 h caused 15.43%, 28.29%, 52.06% apoptotic cells, which was more than that of the untreated group 4.8%, U266 exposure to 10-40 ng/ml TPL for 24 h caused 12.94%, 15.93%,54.04% apoptotic cells, which was more than that of the untreated group 7.76%.In order to explore the possible pathway of the signal conduction of the cell apoptosis induced by TPL. We using western blot to detect the change of caspase-8,-9,-3 and PARP in RPMI 8226 cells. Following treatment with 10-40ng/mI TPL for 12h, typical caspase-9 ,-8, -3 and PARP cleavage were detected in RPMI8226 cells.In order to investigate the possible mechanism and signal conduct pathway of the MM cells apoptosis induced by TPL, the NF-k B activity was assessed by EMSA. RPMI8226 and U266 cells were cultured with TPL (0. 10, 20, 40ng/ml) for 6 hours, and then detected the activity of NF-k B by EMSA. The result showed that as the concentration of drug increased, the activity of NF- k B was down regulated. The inhibit ratio in RPMI8226 was about 17.25%,28.13%,48.13% after culture with 10, 20, 40ng/mI TPL for 6 hours; And the inhibit ratio in U266 cells was 11.76%, 49%, 54.3%...