In Vitro Assessment of Anti-Multiple Myeloma (MM)-Agents On Various MM-Cell Lines (MMCLs) with Use of Bortezomib, Sorafenib, Thalidomide, Lenalidomide and EpiGalloCatechin-3-Gallate (EGCG) Demonstrates to Be a Valuable Tool for the Thorough Analysis and Discovery of Innovative Anti-MM-Agents.
Authors: Udi Josefina , Wider Dagmar , Ihorst Gabriele , Schüler Julia , Wäsch Ralph , Engelhardt Monika 
Published: October 2019

Abstract
Abstract 4922

Introduction
Thalidomide and lenalidomide possess antiangiogenic, antiproliferative, proapoptotic and immunomodulatory effects. The proteasome inhibitor bortezomib induces cell death in MMCLs and has demonstrated synergism on various tumor cell lines, when combined with the multikinase inhibitor sorafenib. Sorafenib targets various kinases involved in tumor growth and angiogenesis, which plays a governing role in numerous cancers, including MM. EGCG, the most active catechin in green tea, has been described to induce anti-MM- and anti-amyloid-, but recently also to prevent bortezomib-induced-effects. We therefore tested these compounds individually and in combinations on 3 MMCLs in order to assess their cytotoxicity, cell growth inhibition and phenotype changes.

Material and Methods
RPMI8226, U266 and L363 were cultured at 1×105 cells/ml, with RPMI1640, 10% FCS and 0.2% pen/strep. On day (d) 0, increasing concentrations of bortezomib, sorafenib, thalidomide, lenalidomide and EGCG were added. On d3 and d6, we determined cytotoxicity and cell viability via trypan blue dye exclusion assay and propidium iodide (PI) staining by flow-cytometry (FACS). Additionally, we analyzed phenotype changes by means of CD138-expression (FACS). To evaluate CD138-expression as well as morphologic changes after sorafenib treatment, we also performed confocal microscopy analyses.

Results
100nM bortezomib showed pronounced cytotoxicity on all 3 MMCLs: mean PI-positivity in L363 was 83.9% on d3, remained stable on d6 and was significantly increased as compared to control-L363-cells (p<0.01). In U266, mean PI-positivity on d3 and d6 was 37.5% and 25.2%, respectively, again being significantly higher than in control-U266-cells (p<0.0001). In RPMI, PI-positivity was similarly increased on d3 and d6 with 94.5% and 91.3%, respectively, again substantially higher than in control-RPMI-cells (p<0.001).
With 10 and 100μM sorafenib in L363, we observed mean PI+ cells on d3 as high as 61.6% and 94.3% and on d6 of 80.8% and 91.8%, respectively (p<0.0001). A statistically significant dose-dependent decrease in viable cells and CD138-expression with 10 and 100μM sorafenib as compared to L363-control-cells could also be detected. By confocal microscopy, CD138-downregulation was prominent, besides manifest morphologic changes. In U266, mean PI+ cells were 33.1% with 10μM and 78.3% with 100μM, again significantly higher than in control-U266-cells (p<0.001), not substantially increasing on d6. Sorafenib's cytotoxicity was likewise evident in RPMI: PI+ cells on d3 with 10 and 100μM were 89.8% and 95.2% (p<0.001), respectively. In contrast, even with 100μg/ml thalidomide, cytotoxicity in L363 cells was subtle, with mean PI+ cells of 10.0% on d3 and 21.6% on d6 (n.s.), with cell viability only slightly decreasing. Thalidomide did not significantly affect U266 cell growth either. Lenalidomide did not increase PI+ cells in L363 on d3, but induced a noticeable PI+-rise on d6 of 19.2% with 10μM (p<0.01) and 15% with 100μM (p<0.001) as compared to control-L363-cells. No statistically significant decrease in viable cells and CD138-expression was observed. With 10 and 100μM lenalidomide in U266, PI+ cells on d6 were higher with 19.9% and 29.6% (p<0.001), respectively, as compared to control-U266-cells. Exposure of all MMCLs to EGCG, with concentrations ranging from 1 to 500μM, showed potent cytotoxic effects, most evident with concentrations of 250μM or higher in L363 (p<0.01).

Conclusions
Bortezomib and sorafenib showed impressive cytotoxic effects as single agents and ongoing experiments suggest additive effects between both compounds, which is currently being investigated, both in vitro and in our in vivo NOD/SCID-IL2-receptor-gamma-chain−/− (NSG)-mouse-model. Further investigations will also validate the recently suggested inhibitory effects of EGCG on bortezomib-induced cytotoxicity. Thalidomide and lenalidomide moderately reduced viable cell numbers, confirming that other mechanisms, such as anti-angiogenesis and immunomodulation are of greater relevance on MM cells. In line with earlier work, EGCG induced a pronounced cytotoxic effect and inhibition of proliferation. Our results demonstrate that our in vivo model is valuable for the thorough analysis and discovery of innovative targeted anti-MM-agents.

Disclosures
No relevant conflicts of interest to declare.