Rice protein prolamin promotes anti-leukemia immunity and inhibits leukemia growth in vivo.

Prolamin is a heat-stable storage protein of rice (Oryza sativa). This study aimed to examine the effect of prolamin on anti-tumor immune response in vitro and leukemia growth in vivo. The prolamin-enriched rice fractions were prepared to stimulate peripheral blood mononuclear cells (MNC) from mice spleen. The MNC-conditioned medium (MNC-CM) was collected to treat leukemia L1210 cells. Human MNC-CM was prepared to treat Jurkat acute T cell leukemia cells. Purified prolamin was orally administered to syngeneic L1210-bearing DBA/2 mice to assess weights of tumor, liver and spleen, liver histopathology, peripheral blood neutrophil count and cytokine levels. Prolamin-prepared MNC-CM inhibited the viability of murine leukemia L1210 cells and human leukemia Jurkat cells, indicating an immunomodulatory effect. In syngeneic L1210-bearing DBA/2 mice, oral administration of purified prolamin dose-dependently decreased the tumor weight and attenuated the leukemia-induced reduction of liver and spleen weights. Prolamin inhibited the increase of peripheral blood leukocyte count. The levels of tumor necrosis factor-α and interferon-γ in MNC-CM and mice serum were significantly increased by prolamin treatment. No significant change in body weight, serum alanine aminotransferase and creatinine levels was noted by prolamin treatment. Rice prolamin could effectively promote anti-tumor immunity and inhibit leukemia growth without significant toxicity.

Involvement of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells.

Leukemia is a type of hematopoietic stem cell malignant cloned disease with high mortality. Cisplatin-based chemotherapy is one of the most common treatments for leukemia. Similar to other chemotherapeutic agents, cisplatin resistance has become a serious issue in cancer therapy. In the present study, we investigated the role of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells. Firstly, the L1210 cell line resistant to cisplatin (L1210/DDP) was established. Compared to its parental cell line, the IC50 value of cisplatin in the L1210/DDP cells was increased 10-fold. Mitofusins (Mfn1 and Mfn2), mitochondrial outer membrane fusion proteins, were markedly upregulated in the L1210/DDP cells, whereas the expression of fission protein Drp1 and inner membrane fusion protein OPA1 were not significantly altered. In addition, mitofusins were also upregulated in the parental L1210 cells subjected to cisplatin stress. To investigate the role of mitochondrial dynamics in the antineoplastic activity of cisplatin, the effect of mitochondrial division inhibitor (Mdivi)-1 on cisplatin­induced cell death, caspase-3 cleavage and ROS production was examined in L1210 cells. We found that 5 µM of Mdivi-1 efficiently attenuated cisplatin-induced cell death, caspase activation and intracellular ROS increase in L1210 cells. Our data indicated that mitochondrial dynamics play an important role in the antineoplastic activity of cisplatin, and mitofusin-mediated mitochondrial fusion may be involved in the process of cisplatin resistance in leukemia cells. Therefore, the present study revealed that mitochondrial dynamics may be a potential target used to improve the antineoplastic activity of cisplatin in leukemia in the future.

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells.

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017.

Comparative studies of oxaliplatin-based platinum(iv) complexes in different in vitro and in vivo tumor models.

Using platinum(iv) prodrugs of clinically established platinum(ii) compounds is a strategy to overcome side effects and acquired resistances. We studied four oxaliplatin-derived platinum(iv) complexes with varying axial ligands in various in vitro and in vivo settings. The ability to interfere with DNA (pUC19) in the presence and absence of a reducing agent (ascorbic acid) was investigated in cell-free experiments. Cytotoxicity was compared under normoxic and hypoxic conditions in monolayer cultures and multicellular spheroids of colon carcinoma cell lines. Effects on the cell cycle were investigated by flow cytometry, and the capacity of inducing apoptosis was confirmed by flow cytometry and Western blotting. The anti-cancer activity of one complex was studied in vivo in immunodeficient and immunocompetent mice, and the platinum levels in various organs and the tumor after treatment were quantified. The results demonstrate that modification of the axial ligands can improve the cytotoxic potency. The complexes are able to interfere with plasmid DNA, which is enhanced by co-incubation with a reducing agent, and cause cell cycle perturbations. At higher concentrations, they induce apoptosis, but generate only low levels of reactive oxygen species. Two of the complexes increase the life span of leukemia (L1210) bearing mice, and one showed effects similar to oxaliplatin in a CT26 solid tumor model, despite the low platinum levels in the tumor. As in the case of oxaliplatin, activity in the latter model depends on an intact immune system. These findings show new perspectives for the development of platinum(iv) prodrugs of the anticancer agent oxaliplatin, combining bioreductive properties and immunogenic aspects.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Preferential enlargement of leukemia cells using cytoskeletal-directed agents and cell cycle growth control parameters to induce sensitivity to low frequency ultrasound.

Sonodynamic therapy (SDT) is a form of ultrasound therapy that has been shown to preferentially damage malignant cells based on the relatively enlarged size and altered cytology of neoplastic cells in comparison to normal cells. This study sought to determine whether cytoskeletal-directed agents that either disrupt (cytochalasin B and vincristine) or rigidify (jasplakinolide and paclitaxel) microfilaments and microtubules, respectively, affect ultrasonic sensitivity. U937 human monocytic leukemia cell populations were treated with each cytoskeletal-directed agent alone, and then sonicated at 23.5 kHz under relatively low power and intensity (20-40 W; 10-20 W/cm(2)), or at 20 kHz using moderate power and intensity (60 W; 80 W/cm(2)). In addition, human leukemia lines U937, THP1, K562, and Molt-4, and the murine leukemia line L1210 were sonicated using pulsed 20 kHz ultrasound (80.6 W; 107.5 W/cm(2)) both with and without the addition of cytoskeletal-directed agents to assess whether cytoskeletal-directed agents can potentiate ultrasonic sensitivity in different leukemia lines. Human hematopoietic stem cells (hHSCs) and leukocytes were sonicated with continuous 23.5 kHz ultrasound (20 W; 10 W/cm(2)) to determine whether this approach elicited the preferential damage of neoplastic cells over normal blood components. To determine whether ultrasonic sensitivity is exclusively dependent on cell size, leukemia cells were also enlarged via alteration of cell growth parameters including serum deprivation and re-addition, and plateau-phase subculturing. Results indicated that cytochalasin B/ultrasound treatments had the highest rates of initial U937 cell damage. The cells enlarged and partially synchronized, either by serum deprivation and re-addition or by plateau-phase subculturing and synchronous release, were not comparably sensitive to ultrasonic destruction based solely on their cell size. In addition, cytochalasin B significantly potentiated the ultrasonic sensitivity of all neoplastic cell lines, but not in normal blood cells, suggesting that preferential damage is attainable with this treatment protocol. Therefore, it is likely that ultrasonic cell lysis depends not only on cell size and type, but also on the specific molecular mechanisms used to induce cell enlargement and their effects on cell integrity. This is supported by the fact that either the microfilament-or microtubule-disrupting agent produced a higher rate of lysis for cells of a given size than the corresponding stabilizing agents.

N-Farnesyloxy-norcantharimide and N-farnesyl-norcantharimide inhibit the progression of leukemia and increase survival days in a syngeneic mouse leukemia model.

This study investigated the anticancer effects of two newly synthesized norcantharidin analogs, N-farnesyloxy-norcantharimide (NOC15) and N-farnesyl-norcantharimide (NC15), in L1210 cells and in a syngeneic mouse leukemia model (L1210 cell line plus DBA/2 mice). We found that the half-maximal inhibitory concentration (IC50) of NOC15 and NC15 on L1210 cells is 1.56 and 2.62 µmol/l, respectively, and that the IC50 of NOC15 and NC15 on human normal lymphoblast is 207.9 and 2569 µmol/l, respectively. In cell cycle analysis, NOC15 could increase the sub-G1 phase, whereas NC15 could induce G2/M arrest. Annexin-V apoptosis assay indicated that both NOC15 and NC15 could induce cell apoptosis. In the syngeneic mouse leukemia model, both NOC15 and NC15 could increase the survival days of mice and decrease the tumor weight. Moreover, both NOC15 and NC15 could retard the increase in peripheral blood leukocyte count due to L1210 cells. In the subcutaneous (s.c.) group, the treatment with NOC15 could retard the decrease in the weight of the liver and the spleen caused by L1210 cells, whereas the treatment with NC15 could retard the decrease in the weight of the spleen caused by L1210 cells. We conclude that the new compounds NOC15 and NC15 have strong anticancer activity and low toxicity both in vitro and in vivo. NOC15 and NC15 may have the potential to be developed into anticancer agents in the future.

Synthesis and anticancer evaluation of benzyloxyurea derivatives.

A series of novel benzyloxyurea derivatives was designed, synthesized by substituting different benzyls or phenyls on N,N'-positions of the hydroxyurea (HU). These target compounds were evaluated for their anticancer activity in vitro against human leukemia cell line K562 and murine leukemia cell line L1210 in comparison with HU by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Some of the compounds showed promising anticancer activity against the cells. Molecular docking experiments with Saccharomyces cerevisiae R1 domain indicated that 4a and 4f' have stronger affinity than 4m and 4n. Flow cytometry study showed that compound 4g exerted greater apoptotic activity against K562 cells line than HU.

Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

Induction of apoptosis associated with chromosomal DNA fragmentation and caspase-3 activation in leukemia L1210 cells by TiO2 nanoparticles.

We investigated the effects of nanosized TiO2 particles on the death of mouse leukemia L1210 cells. TiO2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO2) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO2 particles. TiO2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface.

Deferasirox shows in vitro and in vivo antileukemic effects on murine leukemic cell lines regardless of iron status.

Numerous studies have shown the antiproliferative effect of iron chelating agents (ICAs), which have been used traditionally in patients with secondary iron overload (SIO). Because the in vivo model for these studies has been animals with normal iron status, the antileukemic effect of ICAs in the SIO condition has not been determined clearly. We investigated the in vitro and in vivo effects of ICAs in murine leukemic cell lines regarding the iron status. The viability of both EL4 cells and L1210 cells incubated with either deferoxamine (DFO) or deferasirox (DFX) decreased in a concentration-dependent manner. This effect was most prominent in L1210 cells treated with DFX. The viability of L1210 cells incubated with both ICAs did not change regardless of the presence of ferric chloride. The percentage of apoptosis in L1210 cells treated with DFO or DFX increased in a concentration-dependent manner; however, the expression of Fas showed no significant change. The non-SIO mice and SIO mice bearing L1210 cells showed longer survival than other groups when treated with DFX, whereas the SIO mice treated with DFO showed shorter survival than the control group. The tumor was significantly smaller in the SIO mice treated with DFX or DFO compared with the control group. The iron content of the liver or the tumor in SIO mice decreased after ICA treatment. This study indicates an antileukemic effect of DFX regardless of iron status and suggests that the use of DFX has a survival benefit for SIO leukemia murine model in terms of iron chelation and antileukemic therapy.

Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.

Antitumor effects of synthetic 6,7-annulated-4-substituted indole compounds in L1210 leukemic cells in vitro.

BACKGROUND: Because annulated indoles have almost no representation in the PubChem or MLSMR databases, an unprecedented class of an indole-based library was constructed, using the indole aryne methodology, and screened for antitumor activity. Sixty-six novel 6,7-annulated-4-substituted indole compounds were synthesized, using a strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions, and tested for their effectiveness against murine L1210 tumor cell proliferation in vitro. MATERIALS AND METHODS: Various markers of tumor cell metabolism, DNA degradation, mitotic disruption, cytokinesis and apoptosis were assayed in vitro to evaluate drug cytotoxicity. RESULTS: Most compounds inhibited the metabolic activity of leukemic cells in a time- and concentration-dependent manner but only 9 of them were sufficiently potent to inhibit L1210 tumor cell proliferation by 50% in the low-µM range after 2 (IC(50): 4.5-20.4 µM) and 4 days (0.5-4.0 µM) in culture. However, the antiproliferative compounds that were the most effective at day 4 were not necessarily the most potent at day 2, suggesting different speeds of action. A 3-h treatment with antiproliferative annulated indole was sufficient to inhibit, in a concentration-dependent manner, the rate of DNA synthesis measured in L1210 cells over a 0.5-h period of pulse-labeling with (3)H-thymidine. Four of the antiproliferative compounds had weak DNA-binding activities but one compound reduced the fluorescence of the ethidium bromide-DNA complex by up to 53%, suggesting that some annulated indoles might directly interact with double-stranded DNA to disrupt its integrity and prevent the dye from intercalating into DNA base pairs. However, all 9 antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells, containing (3)H-thymidine-prelabeled DNA, suggesting that these antitumor annulated indoles might trigger an apoptotic pathway of DNA fragmentation. Indeed the antiproliferative annulated indoles caused a time-dependent increase of caspase-3 activity with a peak at 6 h. Interestingly, the compounds with the most potent antiproliferative IC(50) values at day 2 were consistently the most effective at inhibiting DNA synthesis at 3 h and inducing DNA fragmentation at 24 h. After 24-48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of L1210 cells and stimulated the formation of many bi-nucleated cells, multi-nucleated cells, apoptotic cells and micronuclei, suggesting that these antitumor compounds might enhance mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis to induce apoptosis. CONCLUSION: Although annulated indoles may have interesting bioactivity, novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor lead compounds, and investigate their molecular targets and mechanisms of action.

Formulation, antileukemia mechanism, pharmacokinetics, and biodistribution of a novel liposomal emodin.

Emodin is a multifunctional Chinese traditional medicine with poor water solubility. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a pegylated vitamin E derivate. In this study, a novel liposomal-emodin-conjugating TPGS was formulated and compared with methoxypolyethyleneglycol 2000-derivatized distearoyl-phosphatidylethanolamine (mPEG2000-DSPE) liposomal emodin. TPGS improved the encapsulation efficiency and stability of emodin egg phosphatidylcholine/cholesterol liposomes. A high encapsulation efficiency of 95.2% ± 3.0%, particle size of 121.1 ± 44.9 nm, spherical ultrastructure, and sustained in vitro release of TPGS liposomal emodin were observed; these were similar to mPEG2000-DSPE liposomes. Only the zeta potential of -13.1 ± 2.7 mV was significantly different to that for mPEG2000-DSPE liposomes. Compared to mPEG2000-DSPE liposomes, TPGS liposomes improved the cytotoxicity of emodin on leukemia cells by regulating the protein levels of myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein, which was further enhanced by transferrin. TPGS liposomes prolonged the circulation time of emodin in the blood, with the area under the concentration-time curve (AUC) 1.7 times larger than for free emodin and 0.91 times larger than for mPEG2000-DSPE liposomes. In addition, TPGS liposomes showed higher AUC for emodin in the lung and kidney than for mPEG2000-DSPE liposomes, and both liposomes elevated the amount of emodin in the heart. Overall, TPGS is a pegylated agent that could potentially be used to compose a stable liposomal emodin with enhanced therapeutics.

[Part IV. Synthesis and antitumor evaluation of s-triazolothiadiazines and pyrazolo s-triazoles derived from ciproxacin].

An efficient modified route based on the targeting mechanism of antibacterial fluoroquinolones for the shift from the antibacterial activity to the antitumor one was further developed. Using a fused heterocyclic ring, s-triazolothiadiazine as a carboxyl bioisostere of ciprofloxacin, the title compounds, 1-cyclopropyl-6-fluoro-7-piperazin-1-yl-3-(6-substituted-phenyl-7H-[1, 2, 4]triazolo[3, 4-b][1, 3, 4]thiadiazin-3-yl)-quinolin-4(1H)-ones (5a-5e) and their corresponding N-acetyl products (6a-6e), were designed and synthesized, separately. Meaningfully, a ring-contraction of fused six-membered thiadiazine occurred by a sulfur extrusion reaction gave new tri-acetylated fused heterocycles related to pyrazolo[5, 1-c][1, 2, 4] triazoles (7a-7e). The in vitro antitumor activity against L1210, CHO and HL60 cell lines was also evaluated for the synthesized fifteen heterocycles compared to parent ciprofloxacin by methylthiazole trazolium (MTT) assay. Interestingly, the results displayed that fifteen fused heterocyclic compounds showed more significant growth inhibitory activity (IC50 < 25.0 micromo x L(-1)) than that of parent ciprofloxacin (IC50 > 150.0 micromol x L(-1)), and the active order decreased from 7a-7e to 5a-5e to 6a-6e, respective.

Targeting focal adhesion assembly by ethoxyfagaronine prevents lymphoblastic cell adhesion to fibronectin.

BACKGROUND: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V). METHODS: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated by in vitro kinase assay. RESULTS: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of ß1 integrin engagement. Etxfag impaired FN-dependent formation of ß1 clustering without modifying ß1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from ß1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization. CONCLUSION: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.

P-glycoprotein depresses cisplatin sensitivity in L1210 cells by inhibiting cisplatin-induced caspase-3 activation.

Multidrug resistance (MDR) is a phenomenon in which cells become resistant to cytostatic drugs and other substances with diverse chemical structures and cytotoxicity mechanisms. The most often observed molecular mechanism for MDR includes high levels of P-glycoprotein (P-gp)--an ABCB1 member of the ABC drug transporter family. Overexpression of P-gp in neoplastic tissue is an obstacle to chemotherapeutic treatment. Herein, we were focused on differences in apoptosis induced by cisplatin (no substrate for P-gp) between P-gp-positive and P-gp-negative L1210 cells. P-gp-positive cells were obtained by either L1210 cell adaptation to vincristine (R) or L1210 cell transfection with the human gene for P-gp (T) and compared with parental L1210 cells (S). R and T cells were more resistant to CisPt than S cells. R and T cell resistance to CisPt-induced apoptosis could not be reversed by verapamil (a well-known P-gp inhibitor), which excludes P-gp transport activity as a cause of CisPt resistance. CisPt induced a more pronounced entry into apoptosis in S than R and T cells, which was measured using the annexin-V/propidium iodide apoptosis kit. CisPt induced more pronounced caspase-3 activation in S than R and T cells. CisPt did not induce changes in the P-gp protein level for R and T cells. While similar levels of Bax and Bcl-2 proteins were observed in P-gp-negative and P-gp-positive cells, CisPt induced a more significant decrease in Bcl-2 levels for S cells than P-gp-positive cells. Expression of p53 and its molecular chaperone Hsp90 were more pronounced in R and T than S cells. Moreover, CisPt enhanced the upregulation of p53 and Hsp90 in R and T cells to a higher degree than S cells. Apoptosis was shown to be the prevalent mode of cell death in S, R and T cells by the typical DNA fragmentation and cell ultrastructure changes. All of the above findings indicate that P-gp, independent of its drug efflux activity, induced changes in cell regulatory pathways that confer a partial loss of cisplatin sensitivity.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

Bioactivity of synthetic 2-halo-3-aryl-4(3H)-quinazoliniminium halides in L1210 leukemia and SK-BR-3 mammary tumor cells in vitro.

BACKGROUND: Because quinazolines and their derivatives exhibit a wide range of pharmacological profiles, there is a continuous interest among synthetic and medicinal chemists in the discovery of more potent analogs. Ten novel quinazoliniminium salts were synthesized and tested for their effectiveness against murine and human tumor cell proliferation in vitro. MATERIALS AND METHODS: Various markers of tumor cell metabolism, DNA degradation and mitotic disruption were assayed in vitro to evaluate drug cytotoxicity. RESULTS: All compounds induced concentration- and time-dependent antitumor effects in vitro but the 2-chloro-3-(4-methoxyphenyl)quinazolin-4(3H)-iminium chloride (4) was the most effective inhibitor of leukemia L1210 cell proliferation at days 2-4 (IC50: 2.1-0.9 µM), suggesting that the para-methoxyphenyl substituent on the N3 of 4 may enhance the antiproliferative properties of the quinazoliniminium scaffold. In mammary SK-BR-3 tumor cells, 4 reduced the Ki-67 marker of cell proliferation at 24 h and the metabolic activity at days 2 and 4. Moreover, a 1.5- or 3-h treatment with 4 was sufficient to inhibit the rates of DNA, RNA and protein syntheses measured in L1210 cells over 0.5- or 1-h periods of pulse-labeling with 3H-thymidine, 3H-uridine and 3H-leucine, respectively. As 4 did not reduce the fluorescence of the ethidium bromide-DNA complex, this compound was unlikely to directly bind to or destabilize double-stranded DNA. However, 4 induced DNA cleavage at 24 h in L1210 cells containing 3H-thymidine-prelabeled DNA, suggesting that this antitumor drug might trigger an apoptotic pathway of DNA fragmentation. After 12-48 h, 4 weakly increased the mitotic index of L1210 cells but stimulated the formation of many binucleated cells, multinucleated cells and micronuclei, suggesting that this antitumor compound might enhance mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. CONCLUSION: Although 4 may have interesting bioactivity, more compounds based on the quinazoliniminium scaffold must be synthesized to elucidate structure-activity relationships, identify more potent antitumor lead compounds, and investigate their molecular targets and mechanisms of action.

[Heterogeneity of cell population of lymphoma NK/Ly and leukemia L-1210 according to carbohydrate structure of cell surface: immunocytochemical analysis of lectin binding].

The heterogeneity of tumor cell populations according to binding of lectins from lentil (LcL), wheat germs (WGA), peanut (PNA) and concanavalin A was investigated on a model of murine Nemeth-Kellner lymphoma (NK/Ly) and leukemia L-1210. Bound lectins were detected by indirect immunochemical method using home obtained polyclonal antilectin antibodies and immunogold silver staining (IGSS) technique. Significant differences in binding of Con A were revealed between NK/Ly (67% Con A+) and L-1210 (7.2% Con A+) cells, while the differences in binding of other lectins with both types of tumor cells were not significant. A relatively high percentage of PNA+ cells was registered that can indicate a high degree of desialization of membrane glycoproteins.