Danka Cholujova

Realgar (As4S4) nanoparticles and arsenic trioxide (As2O3) induced autophagy and apoptosis in human melanoma cells in vitro.

The aim of the present study was to compare the effect of realgar nanoparticles and arsenic trioxide (ATO) on viability, DNA damage, proliferation, autophagy and apoptosis in the human melanoma cell lines BOWES and A375. The application of various flow cytometric methods for measurements cell viability, DNA cell cycle, mitochondrial potential, lysosomal activity, and intracellular content of glutathione was used. In addition, quantitative PCR, western blotting and multiplex bead array analyses were applied for evaluation of redox stress, autophagic flux, and cell signaling alterations.The results showed that realgar treatment of studied cells caused modulation of cell proliferation, induced a block in G2/M phase of the cell cycle and altered phosphorylation of IκB, Akt, ERK1/2, p38, and JNK kinases, as well as decreased mitochondrial membrane potential. Additionally, it appeared that induction of cell death by both realgar and ATO was dose-dependent, when lower (0.3 µM) dosage increased lysosomal activity and induced autophagy and higher (1.25 µM) concentration resulted in the appearance of apoptosis, while pan-caspase inhibitor attenuated more efficiently realgar- than ATO-induced cell death. Furthermore, low concentrations of ATO and realgar nanoparticles increased the content of intracellular glutathione and elevated γ-H2AX expression confirmed DNA damage preferentially at higher concentrations of both drugs used. Further analysis revealed slight differences in time-dependent phosphorylation pattern due to both realgar and ATO treatments, while significant differences were noticed between cell lines. In conclusion, realgar nanoparticles and ATO treatment induced dose-dependent activation of autophagy and apoptosis in both melanoma cell lines, when autophagy flux was determined at lower drug concentrations and the switch to apoptosis occurred at higher concentrations of both arsenic forms.

Arsenic Sulphide As4S4 Nanoparticles: Physico-Chemical Properties and Anticancer Effects

In this study, arsenic sulphide As4S4 nanoparticles have been prepared, by high-energy wet milling, in the presence of sodium dodecylsulphate, which acts a surfactant. Solid state properties of the nanoparticles were characterised by XRD, Raman scattering, specific surface area and particle size distribution. Changes in surface areas of the particles, in the 0.2 - 5.4 m2 g-1 range, and nanosize distributions, in the 100 - 250 nm range, characterise the surface and morphological properties of nanorealgar. Raman scattering revealed various species in the milled sample that indicate a disproportionate reaction (3As4S4 → 4As2S3 + 4As) occurring as a consequence of milling. Anticancer effects, of the milled species, were confirmed for the human multiple myeloma U266 and OPM1 cell lines. Dissolution experiments in simulated gastric fluid show a possibility for the application of the realgar nanoparticles as an oral dose in future arsenic drug cancer treatments.

A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications.

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

Proteasome inhibition leads to altered signaling in the proteome of cisplatin-resistant human ovarian carcinoma cell line.

To address a precise view into molecular mechanisms of apoptotic signaling pathways after single- or combinatory treatments with specific NF-κB- or proteasome inhibitors and/or cisplatin (CDDP), flow cytometry and western blotting of the cell proteome in human ovarian chemosensitive- and CDDP-resistant cell lines were used. We report here that proteasome inhibition (but not NF-κB inhibition) caused marked alterations in the cell proliferation and cell cycle, as well as in the levels of signaling anti- and pro-apoptotic proteins PARP, NF-κB, IκB-α, Bcl-2, Bax, and lysosome-associated LAMP-1 and ATP-7B molecules in particular proteome fractions. These findings refer to the possibility of regulation of CDDP resistance, inclusive the capacity of lysosomes to export CDDP in certain human ovarian cancer cells by proteasome inhibition.

Realgar nanoparticles versus ATO arsenic compounds induce in vitro and in vivo activity against multiple myeloma

Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As4S4) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti‐MM activity, resulting in significantly decreased tumour burden. The anti‐MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti‐apoptotic proteins. NREA induced G2/M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee‐1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c‐Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem‐like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti‐MM activity when combined with lenalidomide or melphalan. Taken together, the anti‐MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM.