Multiple Myeloma Cells Derived Microvesicles: A Novel Efficient Way To Promote Myeloma Proliferation

Objective Multiple myeloma (MM) is characterized by malignant cloned plasma cells in hematologic cancers, which is still incurable. The pathogenesis of MM remain has not been completely clear. Microversicle (MV) is a product that the cells released through a variety of stress, as cell activation or apoptosis. Tumor-released MV (TMV) as a new kind of mediators, plays a extremely important role in tumor cells'information communication. Moreover it can directly or indirectly supports the tumor cells to escape apoptosis and to promote tumor progression. There are no reports about whether MM-MV exists in MM and what kind of affect it has to MM cell lines. In this reseach, we try to makesuer whether the MM-MV exist in MM cell lines, and then to discuss how do MM-MV impact the MM cells proliferation activity, and what is the molecular mechanism.Methods The multiple myeloma cell lines: RPMI8226 and U266, as the research object, we used electron microscopy, flow cytometry and confocal fluorescence microscopy and other methods to confirm the presence of MM-MV;We used MTT to defect MM cell proliferation, FCM tests MM cell cycles and the expression of Ki67 was tested by Western Blot; To discuss the molecular mechanism that MM-MV effects the MM cell proliferations we use ELISA and Western Blot to investigate the expression of IL-6 , and the activity of NF-KB, AKT, ERK. Results We used lots of experiments to confirmed that MM cells can secret spherical objects which sharp and size is vary , about 100 ~ 1000nm diameter. By testing the most characteristic marker : Annexin-V and CD138, of spherical objects,we confirmed that the expression of Annexin-V and CD138 were 87.2%±3.96% and 76.5%±2.82%, and proofed the spherical objects coming from MM. To study the biological effects of MM-MV in MM cells: in vitro, we confirmed MM-MP can promote MM cell proliferation. MTT investigated: MM cell proliferation of MV groups was significantly stronger than the other groups (P <0.001), in a time and density dependent manner, and the most significant effect chang was in 24h. FCM discovered MM-MV was no significantly changed on the MM cell cycle (P> 0.05). MV groups compared with CON groups of the expression level of Ki67 was increased (P <0.05) at different time points. In vivo, we confirmed: tumor formation time of MM-MV groups was shorter than MM groups (P <0.05), the median of tumor formation time separately were: 15d, 21d in MM-MV groups and MM groups; tumor volumes in nude mice separately were : 14.97 +11.76 mm3, 6.43 + 2.51mm3 in MM- MV groups and MM groups. ELISA confirmed MM-MV increased IL-6 expression level in MM cells. After 6h, 12h, 24h, MV groups were significantly enhanced the expression levels of IL-6 in RPMI8226 cells than CON groups (P <0.01) . After co-cultured U266 cells and MM-MV 6h, 24h ,the MV groups ,compared to the CON groups showed an increased expression of IL-6 levels (P <0.01). Detection of P65, IкB level by Western Blot, we found the expression of IкB was decreased and the expression of P65 was increased in MV groups than CON groups, with significantly difference in 12h, and 24h (P <0.05). To Further exploration the MM-MV promoted MM cells proliferation we test the expression of p-AKT/AKT, p-ERK1/2/ERK1/2 protein. After MM cells treated by MM-MV in 24h , the p-ERK1/2/ERK1/2 and p-AKT/AKT activity were increased in MVgroups compared with CON group in RPMI8226 cells, and the activity of p-ERK1/2/ERK1/2 is marked increased (P < 0.05). They were lower (P <0.05) or slightly increased (P> 0.05) in MV group than CON group in U266 cells. This study confirmed that MM-MV may promote K562, Jurkat, HL-60 and PBMC proliferation, which the effect was significantly in MV from RPMI8226, with statistical significance (P <0.001), and the promoting proliferation effect of MP from U266 had statistically significant in PBMC and Jurkat cells (P <0.001), in K562, HL-60 proliferation is lower (P> 0.05) .Conclusion We confirmed MM-MV exist in the MM cells, and they could significantly increase expression of Ki67, a proliferation protein, then promoted the proliferation of MM cells. MM-MV might be a new way between MM cells for their interaction and a new mechanism for MM disease progression. The effect of MM-MV increasing proliferation may due to the IL-6 secretion, NF-кB activity, ERK and AKT pathways in MM cells. But their specific molecular mechanism remained to be lucubrated.