Hunáková L

HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells

Previously we have reported adipose-tissue derived human mesenchymal stem cells (AT-MSC) as cellular delivery vehicles for tumor-targeted cancer gene therapy. In this report we aimed to determine whether Herpes simplex virus - thymidine kinase (HSV-tk) expressing AT-MSC (TK-MSC) could exert cytotoxic effect on tumor cells upon treatment with prodrug ganciclovir (GCV). Direct co-cultures of human glioblastoma cells 8-MG-BA, 42-MG-BA and U-118 MG with TK-MSC/GCV resulted in substantial viability decrease in vitro. This therapeutic paradigm was most efficient against 8-MG-BA glioblastoma cells exhibiting cytotoxicity (>50%) in the presence of TK-MSC and 0.1microM GCV. Rapid apoptosis induction in three glioblastoma cell lines and TK-MSC demonstrated both bystander cytotoxic effect on tumor cells and GCV conversion-mediated suicide effect on TK-MSC. Furthermore, we were able to demonstrate formation of gap junctions between AT-MSC and human glioblastoma cells as a mechanism contributing to bystander cytotoxicity. Inability of human HeLa and MCF7 to form gap junctions with AT-MSC rendered these cell refractory to the TK-MSC/GCV mediated cytotoxicity. Gap junction intercellular communication (GJIC) capability of AT-MSC with tumor cells further supports the exploitation of mesenchymal stem cells for approaches relying on the bystander effect. Biological consequences of these capabilities remain to be further explored.

Cellular pharmacology of cis and trans pairs of platinum complexes in cisplatin-sensitive and -resistant human ovarian carcinoma cells.

The cellular pharmacology of two pairs of cis and trans platinum complexes has been studied in three human ovarian carcinoma cell lines, a parental relatively cisplatin-sensitive line (CH1), a subline possessing acquired cisplatin resistance (3-fold; CH1cisR) and an intrinsically cisplatin resistant line (13-fold; SKOV-3). Growth inhibition studies showed that both JM335 [trans ammine (cyclohexylaminedichloro dihydroxo) platinum(IV)] and its platinum(II) dichloro homolog JM334 were relatively less cross-resistant against both acquired and intrinsic cisplatin resistant cells. In contrast, resistance circumvention was not apparent in these cell lines with their cis isomeric counterparts (JM149 for JM335 and JM118 for JM334). The trans compound JM335 was more potent than its cis isomer against all three cell lines. There was no clear correlation between intracellular accumulation following 2 h exposure to each compound and resulting DNA platination or growth inhibition. The selective activity of the trans platinum complexes against the SKOV-3 cell line correlated with a deficiency in the repair of adducts within a fragment of the N-ras gene induced by trans compounds whereas adducts induced by the cis counterparts, and cisplatin, were repaired. The CH 1 parental line appeared repair deficient at the gene-specific level to adducts induced by both cis (including cisplatin) and trans compounds. Resistance in CH1cisR was associated with a lack of gene-specific repair of lesions formed by JM118 and JM149. All four compounds induced apoptosis in all three cell lines, as measured by fluorescent microscopy and field inverted gel electrophoresis, although the kinetics of apoptosis was markedly faster for the trans versus cis compounds. In summary, the trans platinum complexes JM335 and JM334 possess unique cellular properties compared to their cis counterparts particularly with respect to gene specific repair of DNA adducts and the rate of induction of apoptosis.

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.

Effects of protein kinase C inhibitor, staurosporine derivative CGP 41 251, on cell cycle, DNA synthesis and drug uptake in neoplastic cell lines.

The protein kinase C inhibitor, staurosporine derivative CGP 41 251, was more efficient than staurosporine in the reversal of decreased anthracycline uptake in the anthracycline-resistant cell subline (A2780/ADR) of ovarian carcinoma. Staurosporine was more efficient than CGP 41 251 in the induction of cytometrically determined DNA fragmentation (cytofluorometric equivalent of apoptosis) in A2780 parental human ovarian carcino-ma cells compared with the drug-resistant A2780/ADR subline and in both human leukemia K-562 cells as well as mouse leukemia L1210 compared with the araC-resistant L1210 cells. Staurosporine was a more potent inhibitor than CGP 41 251 of DNA synthesis in both araC-sensitive and -resistant mouse leukemia L1210 cells. CGP 41 251 was a slightly more efficient inhibitor of thymidlne incorporation than staurosporine in human leukemia K-562 cells and its combination with araC had a higher inhibitory effect on the DNA synthesis in this cell line than staurosporine. CGP 41 251 exerted DNA synthesis inhibitory effects on both araC-sensitive and -resistant L1210 cells. Staurosporine-induced DNA synthesis inhibition in both araC-resistant and -sensitive L1210 mouse leukemia cells was decreased after combined administration with araC.

PROTEIN KINASE INHIBITOR-INDUCED ALTERATIONS OF DRUG UPTAKE, CELL CYCLE AND SURFACE ANTIGEN EXPRESSION IN HUMAN MULTIDRUG-RESISTANT (Pgp AND MRP) PROMYELOCYTIC LEUKEMIA HL-60 CELLS

Protein kinase inhibitors staurosporine and CGP 41251, a benzoylated derivative of staurosporine with selective PKC inhibitory activity, reversed the decreased rhodamine 123 uptake in HL-60/VCR (with Pgp-mediated drug resistance) but not in HL-60/ADR (MRP-mediated drug resistance) cells. CGP 41251 reversed the decreased rhodamine 123 uptake in HL-60/VCR cells more efficiently (when compared on a equimolar basis) than staurosporine. However, the protein tyrosine kinase inhibitor genistein unexpectedly modulated the decreased rhodamine 123 uptake in Pgp positive (HL-60/VCR) cells, but not in HL-60/ADR (MRP positive) cells. Cell surface phenotype of both HL-60 drug-resistant cell sublines was compared with that of the parental, drug-sensitive HL-60 cells. Both drug-resistant cell lines expressed markedly decreased levels of cell surface HLA class I antigen in comparison with the parental HL-60 cells. A similar decreased cell surface expression of HLA class II/DR on both drug-resistant, as well as of CD59 (protectin) on HL-60/ADR cells was found. Both protein kinase C inhibitors studied (staurosporine and CGP 41251) exhibited variable effects on cell surface antigen (HLA, ICAM-1, CD59) expression, suggesting complex interactions between PKC-dependent and -independent mechanisms in the regulation of surface antigen expression in these cell lines. Staurosporine differed from CGP 41251 in the cell cycle alterations induced in the HL-60 cell lines examined. Staurosporine induced the accumulation of cells in the G2/M phase of the cell cycle and the appearance of pre-G0 (apoptotic) cells in both examined drug-resistant cell lines. Staurosporine induced the appearance of cells with high DNA content in HL-60/ADR, but not in HL-60/VCR cells.

Tyrosine kinase inhibitor-induced differentiation of K-562 cells: alterations of cell cycle and cell surface phenotype

Protein tyrosine kinase (PTK) inhibitor herbimycin A inhibited proliferation, induced accumulation of cells in the G0/G1 phase of the cell cycle and a marked increae of hemoglobin-producing human leukemic K-562 cells in vitro. The isoflavonoid PTK- and topoisomerase II inhibitor genistein produced a similar effect with the accumulation of cells in the G2/M phase of cell cycle. Genistein potentiated the effect of herbimycin A on the cell cycle (i.e. decreased the proportion of S-phase cells) and induced an increased proportion of hemoglobin-producing cells. Genistein, but not herbimycin A induced a marked increase in cell surface expression of CD15 (LewisX) antigen. Both of these agents down-regulated CD45 (leukocyte common antigen) and monocyte-associated CD14 antigen on K-562 cells. Neither genistein nor herbimycin A induced increased cell surface expression of glycophorin.

Antitumor activity of benzamide riboside and its combination with cisplatin and staurosporine

Benzamide riboside (BR), a new synthetic nucleoside analogue, has demonstrated a potent cytotoxic activity in murine leukemia in vitro. The purpose of the present investigation was to examine the antitumor activity of BR in mice bearing leukemia L1210. The results revealed that BR possesses a potent antitumor activity in vivo. It increases life-span of mice with leukemia. Synergistic cytotoxicity of BR with select DNA damaging agents, cisplatin (cis-Pt) and staurosporine (STP) was examined in MTT chemosensitivity assay, FACS analyses and apoptotic DNA fragmentation on L1210 cells in culture. A simultaneous treatment of leukemia L1210 cells with the combination of BR and STP resulted in synergistic cytotoxicity that correlated with increased apoptotic activity in those cells. On the other hand, treatment of L1210 cells with combination of BR and cis-Pt resulted in antagonistic cytotoxic effect. Finally, to elucidate the synergistic effect of BR and STP in inducing apoptosis, the attention was directed to the activation of cell death processes through various cell cycle signals. This is the first report describing in vivo antitumor activity of BR and its utilization in combination chemotherapy.

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.

Sulforaphane reduces molecular response to hypoxia in ovarian tumor cells independently of their resistance to chemotherapy

One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1α protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.

Main targets of tetraaza macrocyclic copper complex on L1210 murine leukemia cells

Several metal complex agents have already been introduced into clinical tumor therapy and others are subject of antitumor studies. In this study we focused on the tetraaza macrocyclic copper complex (Cu(TAAB)Cl(2)). We studied the influence of the substance on cell growth, cell cycle, membrane integrity, necrosis, apotosis and glutathione level on the leukemic cell line L1210 in 1-day (22 h) and 3-day (72 h) experiments. The metal complex shows a dose-dependent antiproliferative effect, without affecting cell cycle phases. The present results confirm that copper complex can damage plasmatic membranes and trigger apoptosis, and that after treatment of leukemic cells with the copper complex, glutathione levels were increased.