Striking Anti-Multiple Myeloma (MM) Activity with the In Vitro Use of the Multikinase Inhibitor Sorafenib (S), Cytotoxic Synergy Between Bortezomib (B) and S, but Not Between B and Epigallocatechin-3-Gallate (EGCG; E) Enlarge the Present Knowledge In MM and Promise Novel Insights for Innovative Substance Exploitation In MM-Treatment

As the development of novel anti-MM therapies is pursued worldwide in order to further improve survival in this disease, various innovative agents have to be eagerly tested in in vitro and in vivo models; the former being applied here. Bortezomib (B) has been shown to induce cell death in MMCLs and cytotoxic synergy with sorafenib (S) on various tumor cell lines. S, an oral multikinase inhibitor, targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. EGCG (E), one main green tea constituent, causes MM cell toxicity also, but seems to prevent tumor cell death induced by B in vitro and in vivo, this extend not being fully understood as yet.

RPMI8226, U266 and L363 were cultured with RPMI1640/10% FCS. On day (d) 0, cells were treated with increasing concentrations of B, S and/or E. Cell viability and cytotoxicity were assessed on d3 and d6 via trypan blue and PI-staining. CD138 expression and morphologic changes were evaluated via FACS, immunocytochemistry and confocal microscopy. The effect of S on the chemotactic behaviour of L363 in response to conditioned media (CM = supernatant of M210B4 stromal cells) using 96-well chemotaxis chamber plates was also evaluated. Phosphorylation of ERK1/2 was determined by Western blot. The combined effect of S and B was determined using Calcusyn software: the resulting combination index (CI) defines additive effects (CI=1), synergism (CI<1) and antagonism (CI>1).

With 10 and 100μM S in L363, we observed increased median PI+ cells (62% and 94% on d3, respectively) as compared to the control (median PI+ d0: 11%), with similar increases on d6 (median 81% and 92%, respectively). In line with PI-observations, viable cells and CD138 expression substantially decreased in a dose- and time-dependent manner. After 3 days pre-incubation with increasing S-concentrations, MM cells were stained with Dapi, Phalloidin-Alexa-549 and CD138-FITC and analyzed by confocal microscopy: L363 cells highly expressed CD138 in the absence of S, whereas impressive CD138 downregulation, morphologic changes and reduction of F-actin content were observed with S-concentrations as low as 1μM. L363 cells exhibited a migratory response to CM, whereas after 3 days of preincubation with 10, 20 and 50μM S, L363 cells showed reduced migratory capacity in response to CM. Western blots showed a decrease in p-ERK1/2 expression levels after 24h inbubation of L363 cells with 10μM S. With 100nM B, PI in L363 increased from 11% on d0 to 84% on d3, albeit not as pronounced with 10nM B as was observed with 10μM S. E induced cytotoxicity in L363, particularly with 50 and 100μM, albeit - different to prior reports - B-induced cell death was preserved when the B-E-combination was tested: of note, however, after addition of increasing E-concentrations, no synergism or additive effect, rather than a plateau cytotoxic effect was observed. Combined B and S use showed synergism with 10nM and 10μM, respectively (CI=0.80). MMCLs stably co-expressing fluorescently labelled cytochrome C and histone H2 will allow the detection of induced apoptosis using live-cell imaging after anti-MM agent treatment.

Our MM-based in vitro model revealed that B and S show remarkable therapeutic efficacy as single agents and synergism when combined, which confirms results in other tumor cell lines. E alone induced dose-dependent cell death and decreases in MM cell viability and when combined with B did neither synergize nor abolish B-induced cell death. Our results further enlarge the present knowledge in MM therapy and promise novel insights for innovative substances in the treatment of MM.

No relevant conflicts of interest to declare.