Zosuquidar: An Effective Molecule for Intracellular Ca2+ Measurement in P-gp Positive Cells.

Intracellular calcium, as a second messenger, is involved in multilevel cellular regulatory pathways and plays a role (among other processes) in switching between survival and initiation of cell death in neoplastic cells. The development of multidrug resistance (MDR) in neoplastic cells is associated with the ability of cells to escape programmed cell death, in which dysregulation of intracellular calcium may play an important role. Therefore, reliable monitoring of intracellular calcium levels is necessary. However, such a role might be limited by a real obstacle since several fluorescent intracellular calcium indicators are substrates of membrane ABC drug transporters. For example, Fluo-3/AM is a substrate of P-glycoprotein (ABCB1 member of the ABC family), whose overexpression is the most frequent cause of MDR. The overexpression of ABCB1 prevents MDR cell variants from retaining this tracer in the intracellular space where it is supposed to detect calcium. The solution is to use a proper inhibitor of P-gp efflux activity to ensure the retention of the tracer inside the cells. The present study showed that Zosuquidar and Tariquidar (P-gp inhibitors) are suitable for monitoring intracellular calcium, either by flow cytometry or confocal microscopy, in cells overexpressing P-gp.

Targeting acute myeloid leukemia cells by CD33 receptor-specific MoS2-based nanoconjugates.

Acute myeloid leukemia (AML) is a highly aggressive type of cancer caused by the uncontrolled proliferation of undifferentiated myeloblasts, affecting the bone marrow and blood. Systemic chemotherapy is considered the primary treatment strategy; unfortunately, healthy cells are also affected to a large extent, leading to severe side effects of this treatment. Targeted drug therapies are becoming increasingly popular in modern medicine, as they bypass normal tissues and cells. Two-dimensional MoS2-based nanomaterials have attracted attention in the biomedical field as promising agents for cancer diagnosis and therapy. Cancer cells typically (over)express distinctive cytoplasmic membrane-anchored or -spanning protein-based structures (e.g., receptors, enzymes) that distinguish them from healthy, non-cancerous cells. Targeting cancer cells via tumor-specific markers using MoS2-based nanocarriers loaded with labels or drugs can significantly improve specificity and reduce side effects of such treatment. SKM-1 is an established AML cell line that has been employed in various bio-research applications. However, to date, it has not been used as the subject of studies on selective cancer targeting by inorganic nanomaterials. Here, we demonstrate an efficient targeting of AML cells using MoS2nanoflakes prepared by a facile exfoliation route and functionalized with anti-CD33 antibody that binds to CD33 receptors expressed by SKM-1 cells. Microscopic analyses by confocal laser scanning microscopy supplemented by label-free confocal Raman microscopy proved that (anti-CD33)-MoS2conjugates were present on the cell surface and within SKM-1 cells, presumably having been internalized via CD33-mediated endocytosis. Furthermore, the cellular uptake of SKM-1 specific (anti-CD33)-MoS2conjugates assessed by flow cytometry analysis was significantly higher compared with the cellular uptake of SKM-1 nonspecific (anti-GPC3)-MoS2conjugates. Our results indicate the importance of appropriate functionalization of MoS2nanomaterials by tumor-recognizing elements that significantly increase their specificity and hence suggest the utilization of MoS2-based nanomaterials in the diagnosis and therapy of AML.

Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents.

We established the following two variants of the MOLM-13 human acute myeloid leukemia (AML) cell line: (i) MOLM-13/DAC cells are resistant to 5-aza-2'-deoxycytidine (DAC), and (ii) MOLM-13/AZA are resistant to 5-azacytidine (AZA). Both cell variants were obtained through a six-month selection/adaptation procedure with a stepwise increase in the concentration of either DAC or AZA. MOLM-13/DAC cells are resistant to DAC, and MOLM-13/AZA cells are resistant to AZA (approximately 50-fold and 20-fold, respectively), but cross-resistance of MOLM-13/DAC to AZA and of MOLM-13/AZA to DAC was not detected. By measuring the cell retention of fluorescein-linked annexin V and propidium iodide, we showed an apoptotic mode of death for MOLM-13 cells after treatment with either DAC or AZA, for MOLM-13/DAC cells after treatment with AZA, and for MOLM-13/AZA cells after treatment with DAC. When cells progressed to apoptosis, via JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) assay, we detected a reduction in the mitochondrial membrane potential. Furthermore, we characterized promoter methylation levels for some genes encoding proteins regulating apoptosis and the relation of this methylation to the expression of the respective genes. In addition, we focused on determining the expression levels and activity of intrinsic and extrinsic apoptosis pathway proteins.

Screening of Phenanthroquinolizidine Alkaloid Derivatives for Inducing Cell Death of L1210 Leukemia Cells with Negative and Positive P-glycoprotein Expression.

We describe the screening of a set of cryptopleurine derivatives, namely thienoquinolizidine derivatives and (epi-)benzo analogs with bioactive phenanthroquinolizidine alkaloids that induce cytotoxic effects in the mouse lymphocytic leukemia cell line L1210. We used three variants of L1210 cells: i) parental cells (S) negative for P-glycoprotein (P-gp) expression; ii) P-glycoprotein positive cells (R), obtained by selection with vincristine; iii) P-glycoprotein positive cells (T), obtained by stable transfection with a human gene encoding P-glycoprotein. We identified the most effective derivative 11 with a median lethal concentration of ≈13 µM in all three L1210 cell variants. The analysis of the apoptosis/necrosis induced by derivative 11 revealed that cell death was the result of apoptosis with late apoptosis characteristics. Derivative 11 did not induce a strong alteration in the proportion of cells in the G1, S or G2/M phase of the cell cycle, but a strong increase in the number of S, R and T cells in the subG1 phase was detected. These findings indicated that we identified the most effective inducer of cell death, derivative 11, and this derivative effectively induced cell death in S, R and T cells at similar inhibitory concentrations independent of P-gp expression.

Silver pyridine-2-sulfonate complex - its characterization, DNA binding, topoisomerase I inhibition, antimicrobial and anticancer response.

In the current study the ability of silver pyridine-2-sulfonate complex to exert multiple biological activities is compared with the pharmacological action of silver sulfadiazine (AgSD). Polymeric form of {[Ag(py-2-SO3)]}n (AgPS) was synthesized and characterized by analytical techniques (IR, CHN, TG/DTA, MS) and its molecular formula was established. The crystal structure was determined by X-ray diffraction method and the polymeric complex crystallizes in the triclinic P-1 space group. The stability of Ag(I) complex was verified by 1H and 13C NMR measurements and the interaction with calf thymus DNA through UV-VIS and fluorescence quenching experiments was studied. The Ag(I) complex was able to interact with DNA by dual binding mode: partial intercalation along groove binding. The binding constants were calculated to be in the order of 103 M-1. Topoisomerase I inhibition study have shown that silver complex is inhibiting its activity at concentration of 30 µM. The cytotoxic activity of AgPS and AgSD against mouse leukaemia L1210 S, R and T cell line was also evaluated. AgPS showed higher cytotoxicity than AgSD after 48 h incubation. The results suggest that mechanism of cell death is necrosis with a contribution of late apoptosis. Antimicrobial testing indicates higher growth inhibition effect of AgPS with comparison to commercially available AgSD.

Detection of the Mitochondrial Membrane Potential by the Cationic Dye JC-1 in L1210 Cells with Massive Overexpression of the Plasma Membrane ABCB1 Drug Transporter.

JC-1, a cationic fluorescent dye when added to living cells, is known to be localized exclusively in mitochondria, particularly in good physiological conditions characterized by sufficient mitochondrial membrane potential (ΔΨ). The accumulation of JC-1 in these organelles leads to the formation J-aggregates (with a specific red fluorescence emission maximum at 590 nm), which is in addition to the typical green fluorescence of J-monomers (emission maximum of ∼529 nm). The lack of mitochondrial ΔΨ leads to the depression of JC-1 mitochondrial accumulation and a decrease in J-aggregate formation. Therefore, the ratio between the red and green fluorescence of cells loaded with JC-1 is often used for the detection of the mitochondrial membrane potential. However, JC-1 represents a suitable substrate of the multidrug transporter P-glycoprotein (P-gp). Therefore, the depression of the JC-1 content in intracellular space and particularly in the mitochondria to a level that is inefficient for J-aggregate formation could be expected in P-gp-positive cells. In the current paper, we proved this behavior on parental P-gp-negative L1210 (S) cells and their P-gp-positive variants obtained by either selection with vincristine (R) or transfection with the human gene encoding P-gp (T). P-glycoprotein inhibitors cyclosporine A and verapamil fail to restore JC-1 loading of the R and T cells to an extent similar to that observed in S cells. In contrast, the noncompetitive high affinity P-gp inhibitor tariquidar fully restored JC-1 accumulation and the presence of the typical red fluorescence of J-aggregates. In the presence of tariquidar, measurement of the JC-1 fluorescence revealed similar levels of mitochondrial membrane potential in P-gp-negative (S) and P-gp-positive cells (R and T).