Differential effects of synthesized 2'-oxygenated chalcone derivatives: modulation of human cell cycle phase distribution

Ten structurally related 2'-oxygenated chalcone derivatives, bearing either hydroxy and/or methoxy substituents on the A and B rings, were synthesized through Claisen-Schmidt condensation. The synthesis procedure was relatively easy and had an acceptable yield. The in vitro cytotoxicities of these compounds against the human tumor cells such as Jurkat, U937 cells, and normal cells PHA stimulated PBMCs were investigated. Among those, compounds 1 (IC50 = 2.5 microM), 2 (1.7 microM), and 8 (3.2 microM) showed potent inhibitory activity toward Jurkat cell line. In parallel, compounds 1 (6.7 microM), 2 (1.5 microM), and 10 (5.3 microM) showed the highest activity against U937 cell line. However, the chalcones also inhibit the PHA stimulated PBMCs cells, but the IC50 values were relatively high when compared to the tumor cell line values. Studies were also on the effect of synthesized chalcones on the cell cycle phase distribution. In Jurkat cell line, compounds 7 and 9 showed the highest activity and the most striking effect in reduction of the percentage of cells in the S phase, which was associated with an increase of cells in G2/M phase. In U937 cell line, compound 3 increased the proportion of cells in the G0/G1 phase and reduced the proportion in S phase. In contrast, compounds 1, 9, and 10 showed a decrease effect on the percentage of cells in S phase and an increase effect on the percentage of cells in the G2/M phase of the cell cycle. Whereas in the case of PHA stimulated PBMCs, compounds 1, 4, 8, and 10 increased the percentage of cells in G2/M phase, which was associated with a decrease effect in the S phase of the cell cycle.     

Design of antitumor agents containing carbohydrate based on GLUT1, and evaluation of antiproliferative activity

A series of novel carbohydrate-modified antitumor compounds were designed based on glucose transporter 1 (GLUT1), and evaluated for their anticancer activities against four cancer cell lines. The ribose derivatives (compound 9 and 10) exhibited modest inhibitory activity. The compound 9 significantly inhibited the migration of A549 cell and induced A549 cell apoptosis in a concentration-dependent manner. Moreover, compound 9 blocked A549 cells at the G0/G1 phase. The cellular uptake studies suggested that ribose-modified compound 9 could be taken through GLUT1 in A549 cell line.     

Survivin reduces activation-induced T cell death in G1 phase

A process termed activation-induced cell death (AICD) is responsible for peripheral T cell tolerance after negative selection of self-reactive T cells, and deletion of hyperactivated T cells following the immune response. Cells in G1 phase of the cell cycle are most susceptible to AICD. We have investigated the relationship between the induction of AICD by phorbol 12-myristate 13-acetate plus ionomycin during the cell cycle and the expression of survivin, an inhibitor of the apoptosis protein (LAP) family. AICD was highly induced in cells of the human T cell line Jurkat E6.1 arrested in G1 phase, whereas survivin was hardly expressed in G1 and instead it was highly expressed in G2/M. Moreover, transient over-expression of survivin in G1 partially blocked the induction of AICD. These results suggest that survivin inhibits the induction of AICD, especially in G1 phase.     

Effect of 4-hydroxynonenal on antioxidant capacity and apoptosis induction in Jurkat T cells

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation and may have either physiological or pathological significance regulating cell proliferation. We studied some biochemical effects of HNE, at various concentrations (0.1-100 μM), on Jurkat T cells incubated thereafter for 24, 48 and 72 h. HNE at low concentrations significantly enhanced the proliferation index, whereas at higher concentrations progressively blocked cell proliferation. Caspase 3 activity increased significantly at HNE concentrations between 1 and 10 μM and decreased at higher concentrations. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) increased progressively with HNE concentrations, particularly GSH-Px. Glucose-6-phosphate dehydrogenase (G6PDH) showed a different pattern, increasing at low HNE (1-5 μM) concentrations and rapidly declined thereafter. These results show that HNE may induce growth inhibition of Jurkat T cells and regulate the activity of typical antioxidant enzymes. Furthermore, the protective effect of doubling the foetal calf serum still points out the risk that cultured cells undergo oxidative stress during incubation.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Synthesis, spectral studies and in vitro assessment for antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones

We report here the synthesis, characterization and in vitro antiamoebic activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones (TSC), 1–5, and their bidentate complexes [Ru(η⁴-C₈H₁₂)(TSC)Cl₂] 1a–5a. The biological studies of these compounds were investigated against HK-9 strain of Entamoeba histolytica and the concentration causing 50% cell growth inhibition (IC₅₀) was calculated in the micromolar range. The ligands exhibited antiamoebic activity in the range (2.05–5.29 μM). Screening results indicated that the potencies of the compounds increased by the incorporation of ruthenium(II) in the thiosemicarbazones. The complexes 1a–5a showed antiamoebic activity with an IC₅₀ of 0.61–1.43 μM and were better inhibitors of growth of E. histolytica, based on IC₅₀ values. The most promising among them is Ru(II) complex 2a having 1,2,3,4-tetrahydroquinoline as N⁴ substitution.     

Pathways governing G1/S transition and their response to DNA damage

The ability to self-replicate is a fundamental feature of life, reflected at the cellular level by a highly regulated process initiated in G1 phase via commitment to a round of DNA replication and cell division. Here we briefly highlight recent advances in understanding the molecular pathways which govern the decision of mammalian somatic cells to enter S phase, and the so-called cell cycle checkpoints which guard the G1/S transition and S phase progression against potentially deleterious effects of genotoxic stress. Particular emphasis is put on the emerging parallel yet cooperative pathways of retinoblastoma protein (pRB)–E2F and Myc, their convergence to control the activity of the cyclin-dependent kinase 2 (Cdk2) at the G1/S boundary, as well as the two waves of checkpoint responses at G1/S: the rapid pathway(s) leading to Cdc25A degradation, and the delayed p53–p21 cascade, both silencing the Cdk2 activity upon DNA damage.     

Phenacetin acts as a weak genotoxic compound preferentially in the kidney of DNA repair deficient Xpa mice

Cell cycle checkpoints and DNA repair capacity are critical for the maintenance of genome integrity. We hypothesized that, in comparison to healthy controls, esophageal cancer patients might have a higher frequency of deficiencies in cell cycle checkpoints and/or DNA repair system. Using flow cytometry and comet assay, we assessed the γ-radiation-induced S phase and G2-M phase accumulation, and benzo(a)pyrene-diol-epoxide (BPDE)- and γ-radiation-induced DNA damage, in peripheral blood lymphocytes of 99 newly diagnosed esophageal cancer patients and 112 age-, gender-, and ethnicity-matched healthy controls. The mean γ-radiation-induced cell accumulation at G2-M phase was significantly lower in esophageal cancer patients than the control subjects (case versus control: 5.27% ± 5.11% versus. 7.06% ± 5.04%, P = 0.013). The less G2-M phase cell accumulation resulted in a significant increased risk for esophageal cancer with an odds ratio of 2.08 (95% confidence interval 1.15–3.77). After normalization to baseline S fraction, the radiation-induced increment in the 4N/2N ratio was also significantly lower in esophageal cancer patients than in controls (case versus control: 0.76% versus 1.04%, P = 0.0039). The less increment in the radiation-induced 4N/2N ratio was associated with 2.24(95% confidence interval 1.22–4.11)-fold increase of esophageal cancer risk. We also compared the mutagen-induced DNA damage level among individuals with different S or G2-M phase cell accumulation. We found that the less G2-M phase accumulation was associated with both high BPDE induced and γ-radiation-induced DNA damage in the healthy controls (P for trend = 0.023 and 0.015, respectively). Similar pattern was observed for S phase accumulation (P for trend = 0.033 and 0.022, respectively). However, such association was not seen in esophageal cancer patients. This study provides the first molecular epidemiologic evidence linking increased esophageal cancer risk with defects in cell-cycle checkpoints and DNA repair capacity.     

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity.     

Acceleration of CHO cells into mitosis and reduction of X-ray-induced G2 delay by cordycepin

The mitotic cell selection technique was used to monitor the effect of cordycepin and/or 100 rad of X-rays on the entry of asynchronous or synchronous Chinese hamster ovary cells into mitosis. Continuous exposure of asynchronous cells to 5–50 μg/ml of cordycepin caused a rapid increase in the relative numbers of cells entering mitosis. In irradiated cells, cordycepin also reduced a 120-min mitotic delay by about 80 min and shifted the X-ray transition point about 10 min farther away from mitosis. Further studies showed that synchronous cells, treated continuously with 15 μg/ml of cordycepin starting at mid-to-late S phase, proceeded into mitosis approx. 40 min ahead of controls. This acceleration was associated with a 30-min lengthening of S phase and a reduction in the length of G2 from 80 to about 10 min. Furthermore, cordycepin reduced the 70-min mitotic delay observed for cells irradiated in S phase by 20 min. In contrast to the results for treatment at mid-S phase, continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce by about 60 min the 170-min mitotic delay observed for cells irradiated in G2. Unirradiated synchronous cells treated with cordycepin starting before mid-S did not reach mitosis. Thus, there are the following transition points or intervals for cordycepin: for treatment prior to mid-S phase, cell cycle progression through S is blocked; for treatment between mid-S and late S, progression through S continues but progression through G2 is accelerated; and for treatment during G2, the rate of progression in accelerated only if the cells have been irradiated. These results are discussed in relation to the synthesis during late S and G2 of critical protein molecules essential for mitosis.     

Conformationally restricted PACAP27 analogues incorporating type II/II′ IBTM β-Turn Mimetics. Synthesis, NMR Structure Determination, and Binding Affinity

To probe the importance of a proposed β-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II′ β-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to ¹H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM^10,11]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an -helical conformation within segment T7–L27. For residues S9–R12, our data seem more compatible with a segment of the -helix than with the β-turn previously proposed for this fragment. In compound 1 the -helix, also spanning T7–L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.     

Induction of p53 protein expression by sodium arsenite

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A, Jurkat) and a lymphoblast cell line transformed with Epstein–Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 μM sodium arsenite in Jurkat cells and 10 μM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Selective inhibition of bleomycin-induced G2 cell cycle checkpoint by simaomicin alpha

Human T-cell leukemia-derived Jurkat cells are known to be defective in the G1 checkpoint. DNA-damaging agent bleomycin arrests the cell cycle at G2 phase of Jurkat cells, and microtubule-acting colchicine arrests it at the M phase. Simaomicin alpha, an actinomycete metabolite, itself showed no effect on the cell cycle status of Jurkat cells at least up to 6.0 nM. However, the compound (0.6-6.0 nM) was found to abrogate the bleomycin-induced G2 arrest, yielding a drastic decrease in cells at the G2 phase and increase in cells at the subG1 and G1 phases. On the other hand, the compound did not show any effect on the colchicine-induced M phase arrest in Jurkat cells. Furthermore, the compound showed almost no effect on the cell cycle status of the bleomycin-treated or -untreated normal cell line HUVEC. These data suggested that simaomicin alpha disrupts the cell cycle G2 checkpoint of cancer cells selectively, leading to sensitization of cancer cells to anti-cancer reagents.     

Induction of G1 arrest and apoptosis in human jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27Kip1, p21Cip1/WAF1, and Bax proteins

Pentagalloylglucose, which is found in many medicinal plants, can arrest the cell cycle at G(1) phase through down-regulation of cyclin-dependent kinases 2 and 4 and up-regulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1/WAF1) in human breast cancer cells. Pentagalloylglucose also induces apoptosis in human leukemic cells. However, the mechanisms by which pentagalloylglucose induces these effects is unclear. We now show that pentagalloylglucose inhibits the activities of purified 20 and 26 S proteasomes in vitro, the 26 S proteasome in Jurkat T cell lysates, and chymotrypsin-like activity of the 26 S proteasome in intact Jurkat T cells. The turnover of p27(Kip1) and p21(Cip1/WAF1), which is necessary for cell cycle progression mediated by proteasome degradation, was disrupted by treatment of human Jurkat T cells with pentagalloylglucose. This was shown by cycloheximide treatment and in vivo pulse-chase labeling experiments, and this effect correlated with the arrest of proliferation of Jurkat T cells at G(1). Inhibition of the proteasome by pentagalloylglucose and by the proteasome inhibitor MG132 caused accumulation of ubiquitin-tagged proteins in Jurkat T cells. The addition of pentagalloylglucose to Jurkat T cells enhanced the stability of the proteasome substrate Bax and increased cytochrome c release and apoptosis. Our findings suggest a mechanism for the effect of pentagalloylglucose on the cell cycle in human leukemic cells: that pentagalloylglucose down-regulates proteasome-mediated pathways because it is a proteasome inhibitor.     

Differential regulation of cell cycle-related proteins by CD95 engagement in thymocytes and T cell leukemic cell line, Jurkat

CD95 engagement results in apoptosis in thymocytes and in the Jurkat human leukemic T cell line. Biochemical analyses in CD95-engaged thymocytes and Jurkat cells revealed dysregulation of the G1/S cell cycle control point. Cyclin E was upregulated upon CD95 engagement, suggesting G1-to-S progression, but there was no upregulation of cyclin A. Instead, cyclin E was degraded by caspases. In addition, c-myc that normally acts on S-phase progression through the activation of cdc25A appeared to be involved in the inhibition of S-phase progression after CD95 ligation. This implies that G1-->S progression and apoptosis are intimately linked in cells undergoing CD95 ligation. Furthermore, our data suggest that CD95-induced apoptosis occurs at the G1/S phase transition. We therefore suggest that CD95 engagement not only triggers death signals but also affects the G1/S checkpoint.     

Low-dose anisomycin is sufficient to alter the bio-behaviors of Jurkat T cells

Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. It has been found that a quite low dose of anisomycin is sufficient to block proliferation of primary T lymphocytes. The focus of this study is to explore the possibility of anisomycin to treat human acute leukemia Jurkat T cells in vitro. The results indicated that the low dose of anisomycin could significantly inhibit the colony formation of Jurkat T cells and elevate the inhibition rate of Jurkat T cell growth along with its increasing concentrations. Jurkat T cell cycle was blocked into S-phase by anisomycin. Consistent with the increased proportion of sub-G1 phase, anisomycin promoted Jurkat T cell apoptosis. The CD69 and CD25 expression on the surface of Jurkat T cells was also down-regulated prominently along with the enhancing concentrations of anisomycin, followed by the decreased production of IL-4, IL-10, IL-17, TGF-β and IFN-γ, and the down-regulated expression of phosphorylated-ERK1/2. The results suggest that the suppressive effect of anisomycin on Jurkat T cell growth may be related to inhibiting TGF-β production and ERK1/2 activation, arresting the cell cycle at S-phase and promoting the apoptosis of Jurkat T cells.     

Synthesis,characterization and X-ray structures of new antiproliferative and proapoptotic natural aldehyde thiosemicarbazones and their nickel(II) and copper(II) complexes

Synthesis and characterization of new thiosemicarbazones derived from natural aldehydes (1–9) have been investigated in order to develop a research program aimed at the development of compounds with antiviral, antibacterial, and antitumor properties. These substances contain both a chain with N and S nucleophilic centers with tuberculostatic activity, and an alkyl or terpenic moiety. In addition, a few nickel(II) and copper(II) complexes (10–18), derived also from the previously studied ligands, were synthesized and characterized by means of NMR and IR techniques. The trans-2-octenal N¹-phenylthiosemicarbazone and its nickel complex were also characterized by X-ray diffractometry. Biological studies, performed with some of these compounds, have involved both inhibition of cell proliferation and apoptosis tests in vitro on human leukemia cell line U937 to deepen our knowledge on the way these substances interfere with biological processes in leukemic cells.     

Metal complexes of carboxamidrazone analogs as antitubercular agents. 1. Synthesis,X-ray crystal-structures,spectroscopic properties and antimycobacterial activity against Mycobacterium tuberculosis H(37)Rv

N¹-Benzylidene-pyridine carboxamidrazones and their metal conjugates have emerged as a new class of potential antimycobacterial agents. Nine such carboxamidrazone analogs (L₁–L₉) along with their Cu(II) (MC₁–MC₉) and Fe(III) (MC₁₀–MC₁₈) complexes were synthesized. Single crystal X-ray structures of copper complexes MC₁ and MC₅ were determined which suggest slightly distorted square planer geometries for copper complexes and octahedral geometries for ferric compounds. All compounds were evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv. The results show 32–64-fold enhancement in antitubercular activity upon copper complexation.