Dana Cholujova

Lenalidomide targets clonogenic side population in multiple myeloma: pathophysiologic and clinical implications

Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry-based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.

Induction of Hepatocyte Growth Factor/Scatter Factor by Fibroblast Clustering Directly Promotes Tumor Cell Invasiveness

For determining the malignant behavior of a tumor, paracrine interactions between stromal and cancer cells are crucial. We previously reported that fibroblast clustering induces cyclooxygenase-2 (COX-2), plasminogen activation, and programmed necrosis, all of which were significantly reduced by nonsteroidal anti-inflammatory drugs (NSAID). We have now found that tumor cell-conditioned medium induces similar fibroblast clustering. Activation of the necrotic pathway in clustering fibroblasts, compared with control monolayer cultures, induced a massive >200-fold production of bioactive hepatocyte growth factor/scatter factor (HGF/SF), which made human carcinoma cells spread and invade a collagen lattice. This response occurred only if a functional, properly processed c-Met receptor was present, which was then rapidly phosphorylated. The invasion-promoting activity was inhibited by a neutralizing HGF/SF antibody. NSAIDs, if added early during fibroblast aggregation, inhibited HGF/SF production effectively but had no effect at later stages of cell aggregation. Our results thus provide the first evidence that aggravated progression of tumors with necrotic foci may involve paracrine reciprocal signaling leading to stromal activation by direct cell-cell contact (i.e., nemosis).

Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma.

Background Isothiocyanates, 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 Methods We 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. Results We 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 GSK3α/β 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. Conclusions Our 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.

BACTERIAL GHOSTS AS NOVEL EFFICIENT TARGETING VEHICLES FOR DNA DELIVERY TO THE HUMAN MONOCYTE-DERIVED DENDRITIC CELLS

Summary: Recombinant bacterial ghosts loaded with plasmids were tested as an antigen delivery system and as a potential mediator of maturation for human monocyte-derived dendritic cells (DCs). Bacterial ghosts are cell envelopes derived from Gram-negative bacteria; the intracellular content is released by the controlled expression of plasmid-encoded lysis gene E of PhiX174. All the cell surface structures of the native bacteria, including the outer membrane proteins, adhesins, LPS, lipid A, and peptidoglycans, are preserved. Co-incubation of immature DCs with ghosts resulted in decreased expression of CD1a, CD80, and CD83 molecules, while addition of maturation mix (TNF-α, IL-1β, IL-6, and PGE2) to the cultures enhanced expression of these molecules. No marked changes were observed in the expression of the CD11c, CD40, and CD86 surface molecules. The exposure of DCs to ghosts in combination with maturation mix resulted in a nonsignificant increase in their ability to activate T cells. DCs co-incubated with bacterial ghosts carrying plasmids encoding GFP in combination with maturation mix exhibited high expression levels of GFP (up to 85%). These results indicate that in addition to their well-established use as vaccines, bacterial ghosts can also be used as carriers of nucleic acid-encoded antigens.

Effective gene transfer to melanoma cells using bacterial ghosts

Bacterial ghosts (BG) are cell envelopes preparations of Gram-negative bacteria devoid of cytoplasmic content produced by controlled expression of PhiX174 plasmid-encoded lysis gene E. Eight melanoma cell lines were investigated for their capacity to bind and phagocyte BG derived from Escherichia coli NM522 and Mannheimia haemolytica A23. High capability to bind BG was observed in almost all of the analyzed cell lines, furthermore cells were able to take up BG independently of the used bacterial species. Further, transfection efficiency of BG loaded with DNA in vitro was measured. The Bowes cells exhibited a high expression level of GFP and the incubation of cells with plasmid loaded BG led up to 82% transfection efficiency.

Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.

Modulation of cisplatin sensitivity in human ovarian carcinoma A2780 and SKOV3 cell lines by sulforaphane

Cisplatin resistance is one of the major obstacles in the treatment of ovarian cancer. In an effort to look for new possibilities of how to overcome this difficulty, we studied the mechanisms of the interactions between sulforaphane (SFN) and cisplatin (cisPt) in combined treatment of human ovarian carcinoma A2780 and SKOV3 cell lines. Synergy (A2780) and antagonism (SKOV3) found in MTT assay was confirmed by apoptosis. While SFN significantly potentiated cisPt-induced DNA damage in A2780 cells, it protected SKOV3 cells against cisPt-crosslinking. We revealed a less efficient Nrf-2 pathway inducibility by SFN in A2780 compared to SKOV3 cells, when activation of the Nrf-2 pathway incites its protectivity against cisPt. Thus, different activation of the Nrf-2 pathway may explain the dual effects of SFN.

Increased production of IL-6 and IL-17 in lipopolysaccharide-stimulated peripheral mononuclears from patients with rheumatoid arthritis.

TLR4-mediated inflammatory responses are important for innate immune functions, thus their alterations may participate in the pathogenesis of rheumatoid arthritis (RA). Cortisol is one of the most potent immunomodulatory hormones involved in control of inflammation. In this study, we analyzed TLR4-mediated responses and cortisol effects on the process in peripheral blood mononuclear cells (PBMC) from RA patients. Lipopolysaccharide-stimulated PBMC from 23 female patients and 15 healthy controls were cultured in the presence or absence of cortisol (1 μM) for 24 h. A panel of 17 inflammatory cytokines was analyzed in the cell culture supernatants. Higher (p < 0.05) concentrations of IL-6, IL-17 and MCP-1 were found in lipopolysaccharide-stimulated PBMC from RA patients compared to controls. After normalization of stimulated cytokine secretion to unstimulated cells, a significantly higher (p < 0.05) IL-6 and G-CSF production was found in RA PBMC. Cortisol induced stronger (p < 0.05) suppression of lipopolysaccharide-stimulated secretion of IL-1β, IL-6, IL-17 and G-CSF in RA group compared to controls. The observed higher production of the key inflammatory cytokines by RA PBMC to lipopolysaccharide stimulation supports involvement of TLR4-mediated processes in RA pathogenesis. The higher sensitivity of LPS-stimulated RA PBMC to immunosuppressive effects of cortisol may reflect adaptive processes to chronic inflammation.

Sulforaphane-induced apoptosis involves the type 1 IP3 receptor.

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation.