The Study Of Arsenic Trioxide On Multiple Myeloma Cell Line And Its Mechanisms

Arsenic trioxide (AS2O3) is a novel chemotherapy agent to succeed in treating acute promyelocytic leukemia(APL) firstly in China. The main mechanism of AS2O3 is reported that it can degrade chimeric PML-RAR proteins and induce apoptosis and differentiation in promyelocytes. With more and more insistent study, people find what important is that AS2O3 induces apoptosis not only in APL cells, but also in other malignancies including non- APL leukemia, lymphomas and solid tumors. Recently, Bahlis et al reported AS2O3 has some effect on patients with multiple myeloma ( MM), but the mechanism of action in detail is still unclear. In this study, we examined the effects of AS2O3 on MM cells in order to explore the mechanism of AS2O3 at large and provide a theoretical basis for the clinical treatment against MM.We examined the effects of AS2O3 on the growth of cell line KM3 ( a human MM cell line )by Trypan -Blue dye exclusion. The result showed the growth of KM3 cells was inhibited significantly at the concentration of 0.5~6.0 M AS2O3, and the inhibition rate was increased gradually accompanying with the increase of concentration, which indicated aremarkable dose dependent manner .Apoptosis was detected by morphological study and DNA agarose gel electrophoresis. The apparent difference was detected between KM3 treated cells and untreated cells, we could clearly see the characteristic apoptotic changes in micro-structure, such as cell shrinkage, nuclear atrophy, chromatin condensation roundly, crescent-shaped body and apoptotic body, et al. DNA fragment is confirmed by agarose gel electrophoresis of genomic DNA extracted from KM3 cells treated with 6.0 M AS2O3 for 48 hours, which showed a typical DNA ladder indicated the apoptosis induced by AS2O3.Cell cycle was assayed by flow cytometry (FCM), telomerase activity was assayed by semi-quantitative Telomeric Repeat Amplification Protocol (TRAP); The expression of hTERT ( human telomerase reverse transcriptase ) and TRF2 (telomeric repeated binding factor 2) in transcriptional level were measured by using of semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). With the increased concentration, the result of cell cycle showed GO/G1 phase of KM3 cell treated with AS2O3 for 48 hours decreased gradually corresponding with increased G2/M phase, but S phase varied little compared with control. Cell cycle was arrested at G2/M checkpoint, which indicated the blockage of cell cycle might be one of the mechanisms to induce apoptosis.KM3 cells were treated with different concentrations of AS2O3 (0.5 and l M) for 24 and 48 hours. The result of telomerase activity assay showed a significant decline. These results showed AS2O3 could inhibit telomerase activity. In the presence of different concentration (0,l,2,4,6 M) of AS2O3 for 72 hours, and with the increasedconcentrations of AS2O3, the level of hTERT mRNA, which is an important functional transcriptase of telomerase was gradually declined compared with control, which consisted with the downtrend of telomerase activity. More interestingly, the same decreasing tendency of TRF2 mRNA was observed. Both of them might relate with severity of apoptosis.In conclusion: (1)AS2O3 inhibited the growth of myeloma cell lines significantly in a dose dependent manner. (2) AS2O3 induced apoptosis of KM3 cells, which may be an important mechanism for antitumor of AS2O3 . (3)AS2O3 blocked cell cycle of KM3 cells at G2/M checkpoint. It may be possible that AS2O3 induced apoptosis through the blockage of cell cycle. (4)AS2O3 inhibited telomerase activity through decrease of expression of hTERT mRNA ,which may be one of the mechanism of apoptosis induced by AS2O3 in KM3 cells. (5) AS2O3 declined the expression of TR?2 mRNA during the period of apoptosis induced by AS2O3 in KM3 cells. The expression of hTERT mRNA and TRF2 mRNA declined consistently may be an important parameter to reflect the apoptosis induced by AS2O3 in multiple myeloma cells.