IntroductionMultiple myeloma (MM), a malignancy hallmarked by accumulation of malignant plasma cells in the bone marrow, remains largely incurable despite the use of conventional and novel therapies. 1 The bone marrow (BM) microenvironment promotes tumor cell growth, survival, and confers drug resistance against conventional agents. 2 Although currently available anti-MM strategies have been effective in targeting the bulk of tumor cells, it has been postulated that a tumor-initiating subpopulation or cancer stem cell persists, which may be responsible for eventual relapses. 3 Side population (SP) cells are an enriched source of cancer-initiating cells with stem cell properties, which have been identified in solid tumors, as well as in hematopoietic malignancies. [4][5][6][7][8] The SP cells show a distinct ''low-staining pattern" with the Hoechst 33342 dye. 9 Importantly, SP cells possess the ability to generate non-SP cells both in vitro and in vivo, and are associated with chemoresistance and tumorigenicity in vivo. 4,10 The prevalence and biologic function of SP cells in MM are not fully defined.In the late 1990s, thalidomide was introduced to the treatment of relapsed/refractory MM; however, its effect in patients is associated with dose-and duration-dependent side effects. 11,12 Since then, more potent immunomodulatory drugs (IMiDs), such as lenalidomide, have been introduced. Lenalidomide has been approved for the treatment of both myelodysplasia with deletion of chromosome 5q and for relapsed MM, specifically in combination with dexamethasone. 12,13 Although IMiDs act directly on tumor cells, block adherence to bone marrow stromal cells (BMSCs), modulate angiogenesis and cytokines, and up-regulate host antitumor immunity, the molecular mechanism for their action remains largely undefined, and it is unclear whether they target SP cells in MM. [14][15][16][17][18] In this study, we identified SP cells in MM cell lines as well as in primary MM tumor cells by flow cytometry-based Hoechst 33342 staining, and showed heterogeneity in the percentage of SP cells, as well as the lack of strict correlation between SP fraction and CD138 Ϫ status. SP cells exhibited clonogenic and tumorigenic potential; and importantly, lenalidomide significantly decreased the percentage and clonogenicity of SP cells at clinically relevant concentrations. Moreover, lenalidomide only slightly altered expression of drug-resistant transporter ABCG2 with no effect on functional activity of BCRP1 efflux pump. Modulation of diverse signaling cascades in SP cells by lenalidomide, including changes in Akt, GSK-3␣/␤, MEK1, c-Jun, p53, and p70S6K phosphorylation was observed. Adherence to BMSCs increased the percentage, viability, and proliferation potential of SP cells. Interestingly, both lenalidomide and thalidomide attenuated this stimulatory effect of BMSCs by significantly decreasing SP cell percentages. Therefore, our studies provide insight toward developing novel strategies Submitted February 5, 2010; accepted October 10, 2010. Prepub...

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Dendritic cells (DCs) and natural killer (NK) cells are central components of innate immunity for controlling tumor growth. The therapeutic effects of certain anti-myeloma drugs are partially mediated by targeting the innate immune response. In addition, novel types of natural compounds have been developed that efficiently modulate the activity of both the cellular and humoral compartments of immunity. MGN-3 is known as an activator of natural killer cells, inducer of apoptosis and cytokine production, and modulator of dendritic cell maturation and differentiation in vitro. We have performed a randomized, placebo-controlled study to examine the effects of MGN-3 on innate immune system parameters in 48 multiple myeloma patients. We performed immunophenotypic analysis of peripheral blood samples, determined NK cell activity, and assessed the cytokine profiles of plasma before and during 3 months of treatment. The results demonstrate a clear increase in NK activity in MGN-3-treated patients compared to the placebo group, an increased level of myeloid DCs in peripheral blood, and augmented concentrations of T helper cell type 1-related cytokines. The present study suggests that MGN-3 may represent an immunologically relevant product for activating innate immunity in multiple myeloma patients and warrants further testing to demonstrate clinical efficacy.

Abstract. Most solid tumors display extracellular acidosis, which only partially overlaps with hypoxia and induces distinct adaptive changes leading to aggressive phenotype. Although acidosis is mainly attributable to excessive production of lactic acid, it also involves carbonic anhydrase (CA) IXmediated conversion of CO 2 to an extracellular proton and a bicarbonate ion transported to cytoplasm. CA IX is predominantly expressed in tumors with poor prognosis and its transcription and activity are induced by hypoxia. Here we investigated whether low extracellular pH in absence of hypoxia can influence CA IX expression in cell lines derived from glioblastoma, a tumor type particularly linked with acidosis. Our data show that extracellular acidosis increased the level of CA IX protein, mRNA and the activity of minimal CA9 promoter that contains binding sites for HIF-1 and SP-1 transcription factors. Mutation within each of these two biding sites reduced the promoter activity, but did not eliminate the increase by acidosis. Transfection of HIF-1· cDNA produced additive inducing effect with acidosis. Normoxic acidosis was accompanied by HIF-1· protein accumulation and transiently increased phosphorylation of ERK1/2. Expression of a dominant-negative mutant of ERK2 reduced the CA9 promoter activity in both standard and acidic conditions. Similar result was obtained by inhibitors of MAPK and PI3K pathways, whose combination completely suppressed CA IX expression and abolished induction by acidosis. Altogether, our results suggest that acidosis increases the CA IX expression via a hypoxia-independent mechanism that operates through modulation of the basic CA9 transcriptional machinery.

The online version of this article has a Supplementary Appendix. BackgroundIsothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. Design and MethodsWe evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. ResultsWe observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients' myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G2/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3a/b and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. ConclusionsOur study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.Key words: isothiocyanates, sulforaphane, phenethyl isothiocyanate, PEITC, multiple myeloma, bone marrow microenvironment, signaling pathways. Haematologica 2011;96(8):1170-1179. doi:10.3324/haematol.2010 This is an open-access paper.Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma