| Multiple myeloma (MM) is so far an incurable hematopoietic malignancy. Athough high-dose chemotherapy with autologous bone marrow transplantation has improved the survival significantly, relapse rate remains high, due mostly to existence of residual tumor cells in bone marrow graft. Thus, it is essential to purge myeloma cells from the marrow prior to reinfusion. Recent studys show that alkyl-lysophospholipids (ALP) is a promising agent for purging, since it is selectively toxic to tumor cells and harmless to hematopoietic progenitors. Here in the present study, we used ET-18-OCH3, a member of ALP family which has been widely studyed, added it to culture system of U266 cells or MM bone marrow cells and inverstigated its cytotoxicity against myeloma cells and purging effect of ET-18-OCH3 on bone marrow of MM patients.The aim of our study is to provide experimental data for application of ALP to purge MM bone marrow. It consists of two sections:(1) The antitumor activity of ET-18-OCH3 on U266 cells: U266 cells were incubated with different concentrations of ET-18-OCH3 for some time. These cells were collected, counted and assayed for colony formation ofU266 cells to investigate the cytotoxicity of ET-18-OCH3 on U266 cells. The effect of ET-18-OCH3 in apoptosis induction was determined by the observation of morphology, agarose gel electrophoresis, and flow cytometry. The expression of bcl-2^ c-myc mRNA and proteins were detected by RT-PCR and flow cytometry to study the mechanism of apoptosis. Results showed that ET-18-OCH3 owned significant cytotoxity on U266 cells and inhibitory effect on colonies formation. These effects were time- and dose-dependent. ET-18-OCH3 could also induce time-dependent apoptosis, which was confirmed by morphology characteristics and DNA fragments ladder on electrophoretogram. The apoptosis rate in flow cytometry was 17.53%, with no apoptotic cells found in control. Results of RT-PCR showed marked decrease of bc\-2^ c-myc mRNA by 86% and 72%, respectively. Bcl-2 N c-Myc positive cell percentage decreased by 17% and 60%, respectively.(2) The purging effect of ET-lS-OCHs on bone marrow of MM patients: After incubation with 75 u g/ml ET-18-OCH3 for 4 hours, MM bone marrow cells were collected, analysed for CD38+CD56+CD45"in flow cytometry, measured for IgH CDRIII by semi-nested polymerase chain reaction and assayed for CFU-GM, to evaluate the purging efficacy of ET-18-OCH3 on myeloma cells. To investigate the effect of ET-18-OCH3 on normal hematopoietic progenitors, normal bone marrow cells were assayed for CFU-GM and cytometered for CD34 after incubation with ET-18-OCH3. The expression of bcl-2> c-myc mRNA and proteins were determined by RT-PCR and flow cytometry to study the mechanism of ET-18-OCH3 to purge MM bone marrow ex vivo. Results showed that ET-18-OCH3 was cytotoxic against myeloma cells. The percentage of CD38+CD56+CD45" cells and the expression of IgH CDRIII in MM decreased significantly after treatment with ET-18-OCH3. CFU-GM from MM marrow had little change,indicating that ET-18-OCH3 did not affect the hematopoietic recovery of MM patients. ET-18-OCH3 did not affect normal hematopoietic progenitors, which was supported by the fact that the percentage of CD34+ and CFU-GM from normal bone marrow changed little. Results of RT-PCR showed that the level of both bcl-2 and c-myc mRNA decreased remarkably, as well as Bcl^ c-Myc positive cell percentage.Based on these results, the following conclusions were drawn:(1) ET-18-OCH3 had remarkable cytotoxicity and apoptosis induction effects on U266 cells, with down-regulating the expression of bcl-2 ^ c-myc mRNA and protein as possible mechanism.(2) ET-18-OCH3 had selectively toxic to myeloma cell with sparing of normal hematopoietic progenitors. So it might be used ex vivo bone marrow purging for MM.
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