Target ß-catenin/CD44/Nanog axis in colon cancer cells by certain N'-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides.

Cell surface molecule CD44 plays a major role in regulation of cancer stem cells CSCs on both phenotypic and functional level, however chemical inhibition approach of CD44 to targets CSCs is poorly studied. Herein, we report the discovery of certain N'-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides as a novel inhibitor of CD44. Molecular docking study showed interference of the scaffold of these compounds with ß-catenin/TCF-4 complex, building a direct relationship between CD44 inhibition and observed well-fitted binding domain. Compound 11a, most potent member elicits inhibition effect on TCF/LEF reporter activity conformed the involvement of Wnt pathway inhibition as a mechanism of action. Furthermore, the treatment by the mentioned compound leads to inhibition of embryonic transcriptional factor Nanog but not Sox2 or Oct-4 suggested specific targeted effect. Moreover, the cytotoxicity and cell cycle effect of this series seems to be dependent on CD44 expression.

Targeting HER-2 over expressed breast cancer cells with 2-cyclohexyl-N-[(Z)-(substituted phenyl/furan-2-yl/thiophene-2-yl)methylidene]hydrazinecarbothioamide.

Cyclohexyl thiosemicarbazone derivatives (C1-14) were synthesized, characterized and evaluated against HER-2 over expressed breast cancer cells. The synthesized compounds were screened in vitro against four breast cancer cell lines; SKBr-3, MCF-7, MDA-MB-468 and MDA-MB-231. All the compounds showed activity against HER-2 over expressed SKBr-3 cells with (IC50 = 25.6 ± 0.07 µM-61.6 ± 0.4 µM). The most active compounds inhibit ALDH⁺ breast cancer stem cells more effectively than the cancer stem cells specific agent Salinomycin. Immunohistochemistry staining also confirmed that these compounds inhibit the expression of HER-2 on SKBr-3 cells. Compound C2 significantly inhibited the cell migration and cell adhesion of breast cancer cell lines. Compound C2 was found to most active compound of this series targeting HER-2 over expressed breast cancer cells.

Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.

Curcumin has been shown to possess variety of biological functions including anti-tumor activity. The mechanism by which curcumin inhibit cell proliferation remains poorly understood. In the present report, we investigated the effect of curcumin on the activation of apoptotic pathway in T-cell acute lymphoblastic leukemia (T-ALL) malignant cells. Our data demonstrate that curcumin causes dose dependent suppression of proliferation in several T cell lines. Curcumin treatment causes the de-phosphorylation/inactivation of constitutively active AKT, FOXO transcription factor and GSK3. Curcumin also induces release of cytochrome c accompanied by activation of caspase-3 and PARP cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases, prevents caspase-3 activation and abrogates cell death induced by curcumin treatment. Finally, treatment of T-ALL cells with curcumin down-regulated the expression of inhibitor of apoptosis protein (IAPs). Taken together, our finding suggest that curcumin suppresses constitutively activated targets of PI3'-kinase (AKT, FOXO and GSK3) in T cells leading to the inhibition of proliferation and induction of caspase-dependent apoptosis.

A nuclear protein tyrosine phosphatase induces shortening of G1 phase and increase in c-Myc protein level.

PTP-S2 is a ubiquitously expressed nuclear protein tyrosine phosphatase which shows increased expression upon mitogenic stimulation in a variety of cells in vitro and in vivo. In order to understand the role of this enzyme in cell cycle progression, tetracycline-regulated HeLa clones expressing PTP-S2 were isolated and characterized. Tetracycline-controlled expression of PTP-S2 increased the rate of cell proliferation. An analysis of the distribution of cells in various phases of the cell cycle in an exponentially growing cell population showed that there was a large decrease in the percentage of cells in G1 phase in a PTP-S2-expressing population of cells compared to nonexpressing cells. This decrease in the percentage of cells in G1 was dependent on the level of PTP-S2 expression. There was a corresponding increase in the percentage of cells in G2/M but no significant increase in the percentage of cells in S phase. An analysis of the time course of cell cycle progression after release from double thymidine block showed that the duration of G1 phase was significantly shortened in cells induced to express exogenous PTP-S2. However, the duration of S phase was not significantly altered and the duration of G2 phase was increased to some extent. Induction of PTP-S2 expression was associated with an increase in c-Myc protein levels, although the c-Myc mRNA level was not changed. Our results suggest that overexpression of PTP-S2 promotes progression of cells through G1 to S phase and is associated with increased level of c-Myc protein through a posttranscriptional mechanism.

Modulation of alpha5beta1 integrin functions by the phospholipid and cholesterol contents of cell membranes.

Several modifications of the alpha5beta1 integrin, which alter its intracellular and extracellular interaction with fibronectin and other proteins, have been reported. However, the significance of the lateral mobility of integrin molecules in the plasma membrane, as a regulator of their distribution and function, is poorly understood. We examined this problem by increasing the cholesterol content of plasma membranes, and consequently modifying the fluidity of membrane phospholipids, in rat fibroblasts. Under these conditions, the clustering of alpha5beta1 integrin molecules in focal adhesions, their adhesion to the cell-binding domain of fibronectin, and their association with the cytoskeletal protein talin were significantly enhanced as compared to control cells. However, the activation of MAP-kinase pathways by the association of fibronectin with alpha5beta1 integrin, and its association with integrin-linked kinase (ilk), were suppressed. The treated cells also showed distinct changes in shape, and their actin stress fiber network was more dense and thick as compared to control cells. The changes in fluidity of phospholipids occurred differentially and fluidity of phosphatidyl-ethanolamine increased, while that of phosphatidyl-choline was reduced. Our results suggest that proteins in focal adhesions could be partitioned in specific lipid domains, which regulate specific aspects of alpha5beta1 integrin functions.

Cellular radiosensitivity, radioresistant DNA synthesis, and defect in radioinduction of p53 in fibroblasts from atherosclerosis patients.

Earlier studies have suggested that both cancer and atherosclerosis may follow a common pathway in the early stage of development and share certain risk factors. One report indicated that the gene responsible for the radiosensitive, cancer-prone, multisystem disorder ataxia telangiectasia (AT) may increase the risk of developing ischemic heart disease. The present studies were carried out to find similarities, if any, between atherosclerosis patients and AT homozygotes or heterozygotes (ATHs) in their cellular/molecular response to ionizing radiation, which acts as a carcinogen as well as an atherogen. Fibroblast cell strains developed from healthy subjects and from AT homozygotes, ATHs, and atherosclerosis patients were compared for (1) survival, by the colony-forming assay and (2) DNA synthesis inhibition after irradiation, determined by [3H]thymidine incorporation, cell cycle distribution, and the expression of p53 and p21 proteins, analyzed by flow cytometry. Fibroblasts from the atherosclerosis patients as a group, compared with the healthy subjects, showed enhanced sensitivity to chronic (low-dose-rate) irradiation. A majority of the cell strains representing atherosclerosis patients exhibited varying degrees of radioresistant DNA synthesis (RDS), with roughly 33% showing an AT-like and the rest an ATH-like response. All cell strains with an AT-like and one quarter with an ATH-like RDS were found to be defective in the radioinduction of both p53 and p21 proteins, which are concerned with cell cycle regulation. An absence of G1 arrest after irradiation was observed in cell strains lacking a radioinduced expression of p53 and p21. Cellular/molecular defects leading to increased radiosensitivity, reduced induction of p53/p21, and cell cycle deregulation found to be associated with cancer-prone disorders such as AT may constitute important risk factors for atherosclerosis as well.

A Ca(2+)-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry.

When freshly starved amoebae of Dictyostelium discoideum are loaded with the Ca(2+)-specific dye indo-1/ AM and analyzed in a fluorescence-activated cell sorter, they exhibit a quasi-bimodal distribution of fluorescence. This permits a separation of the population into two classes: H, or "high Ca(2+)-indo-1 fluorescence," and L, or "low Ca(2+)-indo-1 fluorescence." Simultaneous monitoring of Ca(2+)-indo-1 and Ca(2+)-chlortetracycline fluorescence shows that by and large the same cells tend to have high (or low) levels of both cytoplasmic and sequestered Ca2+. Next we label H cells with tetramethylrhodamine isothiocyanate (TRITC) and mix them in a 1:4 ratio with L cells. In the slugs that result, TRITC fluorescence is confined mainly to the anterior prestalk region. This implies that amoebae with relatively high Ca2+ at the vegetative stage tend to develop into prestalk cells and those with low Ca2+ into prespores. Polysphondylium violaceum, a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca(2+)-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azhar et al., Curr. Sci. 68, 337-342 (1995)), a prestalk-prespore difference in cellular Ca2+ is present in the cells of the slug in vivo. These findings are discussed in light of the possible roles of Ca2+ for cell differentiation in D. discoideum.

Modulation of alpha 5 beta 1 and alpha V beta 3 integrins on the cell surface during mitosis.

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of alpha 5 beta 1 and alpha V beta 3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of alpha V beta 3 and very low levels of alpha 5 beta 1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide -GRGDSP-, causes an increase in the intracellular levels of Ca2+ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, alpha 5 beta 1 and alpha V beta 3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase.

Flow cytometric study of changes in the intracellular free calcium during the cell cycle.

We measured the intracellular levels of free cytoplasmic calcium in different phases of the cell cycle in viable rat fibroblasts, using two parameter flow cytometric analysis with Hoechst 33342 as the DNA specific dye and Fluo-3 as the calcium sensitive dye. We studied changes in calcium levels during the G1 phase of cell cycle by arresting cells with chemical agents such as staurosporine and hydroxyurea or by density dependent arrest of cell growth. We show that levels of calcium are lowest at the beginning of G1 phase but rise steadily with its progression and culminate at the G1/S border. Our results suggest that complex changes occur in calcium levels during the process of mitotic division. During progression of S and G2 phases, calcium levels decline and increase respectively. Our results offer a new methodology to estimate intracellular calcium levels in specific phases of the cell cycle. Based upon these results we propose a general scheme representing the changes in the intracellular calcium concentration during the progression of the cell cycle.

Apoptosis: the in vivo mechanism which mediates spontaneous AK-5 tumor regression.

We have studied the mechanism of induction of apoptosis in a rat histiocytoma in vivo. Tumor cells are killed by necrosis and apoptosis when injected into immune animals; however, naive animals are incapable of killing the tumor cells. Tumor cells show DNA fragmentation within 3 h after transplantation in the peritoneal cavity. Natural killer (NK) cells act as the effector in causing tumor cell death through apoptosis since animals depleted of their NK cell population did not show target cell DNA fragmentation. Herbimycin A inhibits the induction of apoptosis, suggesting the requirement for protein phosphorylation.

Flow cytometric measurement of NK cell immunoconjugates by pulse width processing.

Pulse width analysis, in flow cytometry, has been widely used for optimal cell size resolution in cell kinetics analysis. Pulses, generated by scattered light or fluorescence of cells, are electronically analyzed for their height and width. The information generated from these two properties of the pulses is utilized to distinguish signals from single cells vs. signals from cell clumps or aggregates. Pulse width, unlike pulse height, is more sensitive to differences in cell diameter, and therefore can discriminate very small differences in it, which pulse height cannot. We have exploited this property of pulse widths to measure immunoconjugates between NK cells and their targets. Discrimination of the free target cells from the conjugated ones is possible by the pulse widths of only light scatter signals, both forward and/or orthogonal. This resolution was not obtained if pulse height of the same signals was visualized. Using this resolution it was possible to distinguish single cells from the aggregates between target and effector cells. We propose that this is a better method for distinguishing conjugates than the method in which prior vital staining of cells is used.

Conjugate formation between effector and target as a function of cytotoxicity in antibody-dependent killing of AK-5 tumor.

The present study was carried out to mechanistically view a possible correlation between the process of, conjugate formation and its relation to target cell death. AK-5 killing is mediated by CD8+ natural killer cells through ADCC. Immune effectors on exposure to antibody primed AK-5 tumor cells formed tight conjugates. Ability of various cell types (NK, T, monocytes and macrophages) to form conjugates was evaluated. Marked increase in the number of NK cells binding to the target as compared to the other cell types was observed. Cytotoxicity of free and bound effectors against antibody tagged AK-5 cells demonstrated a reduced cytotoxic ability of the former in contrast to a significantly high lytic potential of the bound effectors. The results highlight the requirement for priming of NK cells which mediate killing of AK-5 tumor and provide additional evidence that formation of stable conjugates acts as the first signal for triggering lymphocyte activation and effective target cell lysis.

The level of sequestered calcium in vegetative amoebae of Dictyostelium discoideum can predict post-aggregative cell fate.

When freshly starved amoebae of Dictyostelium discoideum are stained with chlortetracycline (CTC), a cell type-specific fluorescent probe for membrane-associated calcium (CA2+) the resulting fluorescence distribution falls into two functional classes. Fluorescence-activated cell sorting shows that highly fluorescing amoebae tend to enter the prestalk pathway while those with low fluorescence tend to become prespores. In the light of previous findings, these results indicate that in addition to cell cycle phase at starvation, phenotypic variation in the level of sequestered calcium is an early correlate of cell fate.

Heterogeneity in DNA content & proliferative status of human brain tumours.

Intra-tumour and inter-tumour heterogeneity in the cytokinetic organization was studied in 235 primary human brain tumours. DNA index (DI; relative tumour cell DNA content) and proliferating fraction (%PF; a measure of proliferative status) were analyzed in tumour biopsy by flow cytometry using a DNA specific fluorochrome (DAPI) and internal standards (chicken erythrocytes, CE). Incidence of micronuclei was studied in tumour biopsy tissue as well as in explants maintained in organ culture. Clonal diversity (implied by the presence of multiple peaks in the DNA histograms) was highest among medulloblastomas (44%) followed by gliomas (19%) and meningiomas (14%). Nearly 85 per cent of the malignant gliomas analyzed (histological grade III/IV) exhibited a great deal of regional variation in the proliferative status as well as micronuclei frequency as compared to meningiomas. Inter-tumoural variations in the DNA content was highest among gliomas (0.9 < DI < 3.6) and lowest among schwannomas (1.7 < DI < 2.2). Similarly, the distribution of %PF values was also broader (10-49%) in gliomas as compared to the other primary brain tumours (5-36%). Analysis of tumours taking both DI and %PF values improved the ability to discern histologically graded low and high tumours. Analysis of clonal diversity and spatial heterogeneity in the cytokinetic parameters could complement the clinicopathological findings in assessing the biological behaviour of human brain tumours, facilitating the prognostification and design of otpimal treatment regimen.

Status of different immune cells during regression of a rat histiocytoma.

Status of CD4 and CD8 positive T-lymphocytes, natural killer cells and macrophages has been studied in animals which reject AK-5 histiocytoma. Analysis of immune cells which enter the tumour showed higher number of CD8 positive and NK cells around day 25 after tumour transplantation, suggesting their participation in tumour rejection.

Identification of a marker chromosome in a rat ascitic tumor by flow cytometry using a modified method for preparing chromosome suspensions.

Preparation of good chromosome suspensions is a crucial step in obtaining good flow karyotypes from different types of cells. Although several methods are in use, not all are suitable for all cell types. Primary cells from murine tissues are specially difficult to flow karyotype. We have modified the existing protocols for preparing chromosome suspensions from the Zajdela ascitic hepatoma (ZAH) cells of rat and have constructed their flow karyograms. Our karyograms show a distinct marker chromosome in these cells, which could not be resolved using other protocols. We have also used this method for preparing chromosomes from the bone marrow cells of the rat and have compared their flow karyograms with the ZAH karyotype. We expect that our protocol will be useful for studying other types of tumor and normal cells.