Sulforaphane and PEITC Augment Activity of Conventional and Novel Anti-Myeloma Drugs

Abstract

Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.