Inhibitors of protein kinase C sensitise multiple myeloma cells to common genotoxic drugs

Objectives: Despite high dose treatment regimes multiple myeloma (MM) disease is still not curable. Patients become resistant to cytotoxic drugs and die of disease progression. Therefore, besides new cytotoxic compounds drug sensitisers are urgently needed. Methods: The MM cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the common anti-myeloma drugs like melphalan together with protein kinase C (PKC) inhibitors. Growth inhibition was measured using the water-soluble tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay), and apoptosis was determined by flow cytometry after staining with fluorescein isothiocyanate-labeled annexin V (annexin-V-FITC) and propidium iodide. Intracellular signalling was shown by western blotting. Results: In this study we show that the combination of melphalan or doxorubicin with a PKC inhibitor, Go6976 or enzastaurin, strongly increases cell toxicity. Increase of cytotoxicity is shown to be due to increased induction of apoptosis. Furthermore, we show that the protective effect of human bone marrow stromal cells (hBMSC) is abrogated by the PKC inhibitors. Finally, western blotting experiments revealed that incubation of myeloma cells with cytotoxic drugs like melphalan or doxorubicin leads to increased phosphorylation and therefore degradation of inhibitor of nuclear factor kappa B (I kappa B) and release of nuclear factor kappa B (NF kappa B). In contrast, enzastaurin inhibits phosphorylation of I kappa B. Conclusions: We conclude that the combination of conventional drugs and PKC inhibitors might be very effective and represents a new strategy in the treatment of MM.