Antitumor Activity Of Fludarabine Against Human Multiple Myeloma In Vitro And In Vivo

Multiple myeloma (MM) is characterized by excess plasma cells in the bone marrow (BM) associated with monoclonal immunoglobulin (Ig), with clinical manifestations including lytic bone lesions, anaemia, renal failure, immune compromise, and hypercalcaemia. MM remains largely incurable despite conventional and high-dose chemotherapies. Fludarabine is an adenine nucleoside analog. F-ara-ATP has multiple mechanisms of action, which are mostly directed toward DNA. These include inhibition of ribonucleotide reductase, incorporation into DNA resulting in repression of further DNA polymerisation, and inhibition of DNA ligase and DNA primase. Collectively, these actions affect DNA synthesis, which is the major mechanism of F-ara-A-induced cytotoxicity. Secondarily, incorporation into RNA and inhibition of transcription has been shown in tumor cell lines. At present, fludarabine has been applied in hematological malignancy, but there was a few report of the anti-tumor potential and mechanism of fludarabine in multiple myeloma. In this study, we investigated the effects of fludarabine on MM cell lines, and studied the mechanisms of cytotoxicity of fludarabine in myeloma cells with attention to the alteration of survival pathways, including Akt and the inhibitor of apoptosis proteins (IAP) family. Furthermore, we also evaluated the potent antitumor activity of fludarabine in human myeloma RPMI8226 xenograft mice model.The effect of fludarabine on growth of MM cell lines was determined using MTT assays. Fludarabine inhibited efficiently the proliferation of RPMI8226 cells in a dose-time-dependent fashion, with 50% inhibition (IC50) at 24 h of 1.54μg/ml (Fig. 1A). The IC50 of MM.1S and MM.1R cells at 48 h was 13.48μg/ml and 33.79μg/ml, respectively. In contrast, U266 cells were resistant to fludarabine Because IL-6 acts as a growth factor for MM cells, we evaluated the effect of fludarabine on RPMI 8226 in the presence of exogenous IL-6.IL-6(50ng/ml) did not provide protection against fludarabine-induced growth inhibition and apoptosis. We next examined whether fludarabine could enhance the effect of dexamethasone on MM1.S cell line. The results showed that fludarabine enhanced significantly the effect of dexamethasone. To explore the possible mechanism of the inhibition of fludarabine on MM cells, further investigation was carried out from the three stratifications of inducing apoptosis and cell signaling pathway.In order to exactly analyze the cell apoptosis quantitatively and qualitatively, the flow cytometry was also used to evaluate percentage of hypodiploid, translocation of phosphatidyl serine (PS) and cell cycle. We also investigated advanced stage apoptosis by TUNEL assay. It showed that RPMI8226 following treatment with 2μg/ml fludarabine for 6h, 12h, and 24h, a progressive increase in sub-G0/G1 phase cells; and apoptosis induced by fludarabine was also confirmed using FACS analysis following annexinV-PI staining. RPMI8226 exposure to 1-4μg/ml fludarabine for 24 h caused 8.84%, 12.25%, 21.71%, 63.28%, and 65.94% apoptotic cells, which was more than that of the untreated group (4.13%).The result of TUNEL also display the rate of positive cell were correlated with the time of RPMI8226 treatment with fludarabine.In order to explore the possible signal pathway of apoptosis induced by fludarabine, we used western blot to detect the activation of caspase-7,-8,-9,-3 and PARP in RPMI 8226 cells. Following treatment with 2μg/ml fludarabine for 6h, 12h, and 24h, typical caspase-9,-8,-7,-3 and PARP cleavage were detected in RPMI8226 cells.We further studied the function of intrinsic pathway in apoptosis induced by fludarabine.The results showed RPMI8226 following treatment with 2μg/ml fludarabine for 6h, 12h, and 24h, mitochondria membrane potential manifestly descreased, the smac and bax protein expression increased slightly, in contrast the bid expression level was down-regulated significantly.Next, we used western blot to detect the change of Akt and IAPs in MM cells. It showed that the xIAP and Survivin expression descreased slightly in RPMI8226 following treatment with 2μg/ml fludarabine for 6h, 12h, and 24h. In contrast, the cIAP expression level had no change. Four kinds of MM cells exposure to 2μg/ml fludarabine for 24h, the survivin expression descreased in MM.1R, MM.1S, and RPMI8226, but not U266 cells. Among four kinds MM cells, the expression of p-AKT was highest in RPMI8226, MM.1R, and MM.1S. The lowest was U266. Fludarabine inhibited phosphorylation of Akt in some, but not all myeloma cell lines, reflecting a high degree of heterogeneity. We therefore evaluated the contribution of this pathway in fludarabine-mediated apoptosis. Apoptosis assays were done on myeloma cell lines treated with the Akt inhibitor LY294002 in the presence or absence of fludarabine, LY294002 slightly induced the apoptosis of RPMI8226 and not of U266 cells. Interestingly, pronounced apoptosis of MM.1S and MM.1R cells were observed. Slight potentiation was observed in all of tested cell lines except U266 when LY294002 and fludarabine were added together.Fludarabine also showed a significant antitumor activity in vivo. It cound inhibit the growth of tumor, and TLTNEL assay showed an evident increase in apoptotic nuclei in RPMI8226 tumors treated with 40 mg/kg fludarabine at day 10.Summary:(1) Fludarabine inhibited the growth of MM cell lines in dose-dependent manners. Exogenous IL-6 could not overcome the growth inhibition induced by fludarabine.(2) Fludarabine enhanced significantly the effect of dexamethasone.(3) Treatment with fludarabine induced RPMI8226 cell apoptosis in dose- and time-dependent manners.(4) Mechanism of fludarabine-induced apoptosis of MM cell involved in①Activition of caspase -7, -8, -9, -3 and PARP;②Down regulation of bid and smac; up regulation of bax;③Fludarabine decreased the level of surviving;④Fludarabine decreased the level of p-Akt. (5) Fludarabine also showed a significant antitumor activity in vivo.(6) Fludarabine may be a new choose for the treatment of MM patients.