The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 muM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4',5-trimethoxy-3'-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.

Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells.

According to the method used in our laboratory,our group synthesized (DIPP-Trp)2-Lys-OCH 3. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC 50 of 15.12 and 42.23 microM, respectively. (DIPP-Trp) 2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells;the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter,USA). Phosphatidylserine could signi?cantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells.The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining.It was concluded that (DIPP-Trp) 2-Lys-OCH3 not only induced cells to enter into apoptosis,but also affected the progress of the cell cycle.It may have arrested the K562 and HeLa cells in the G 2/M,S phases,respectively.The apoptotic pathway was pulsed at this point,resulting in the treated cells entering into programmed cell death.(DIPP- Trp)-Lys-OCH is a potential anticancer drug that intervenes in the signalling pathway.

Altered remodeling of nucleolar machineries in cultured hepatocytes treated with thioacetamide

Thioacetamide (TA) is converted into a hyperacetylating agent which causes hepatic necrosis, regeneration, cirrhosis and cancerous transformation. One of the most characteristic toxicities of TA in rat is observed with a 50 mg/kg per day which induces nucleolar enlargement different from that in regenerating liver. From TA-treated liver, the nucleoli were isolated and characterized for an altered nucleolar signal transduction system. Immunochemistry revealed that the poisoned nucleoli had increased levels of both nucleolus specific proteins (nucleophosmin and nucleolin) and various signal molecules (CK2, Erk1/2, p38, protein kinases A and C, and cyclin A). Using flow cytometry, the nucleoli were found to be in G2-arrested nuclei. Manifestation of the nucleolar enlargement could be readily observed using an ex vivo hepatocyte culture. There were two types of nucleolar enlargement. One was observed in normal hepatocytes with light density of enlarged nucleoli. The other was in TA-treated hepatocytes with dense and compact density of enlarged nucleoli, which contained a 3 to 5-fold higher nudeophosmin content than the control. In vitro induction of nucleolar enlargement with TA was possible. As soon as the hepatocytes anchored on a collagen coat, exogeneous TA (higher than 1 microg/mL) could induce dense and compact nucleoli. However, when an exogeneous drug was added after monolayer formation (1 day), no drug-induced nucleolar enlargement was observed.

Avaliaçäo da fase gama 2 em amálgamas com diferentes tempos de escultura / Y2 phase volumetric evaluation in amalgams with time crystallization differents

Determinou-se o percentual volumétrico de fase y2 através do método colorimétrico quantitativo e o tempo de cristalizaçäo, em três ligas para amálgama de alto teor de cobre, PRATIC NG2, PERMITE C (rápida, regular e lento) e DISPERSALLOY. As ligas comerciais selecionadas apresentavam-se todas envasadas em cápsulas. As ligas Permite-C continham quantidades de liga/Hg onde a proporçäo variava de 1/0,86 a 1/0,96 alterando desta forma a velocidade de reaçäo do amálgama com tempo de cristalizaçäo rápido, regular e lento, o que permitia ao operador a opçäo de tempos variados para a elaboraçäo da restauraçäo. Para determinaçäo da fase y2 foram confeccionados 10 corpos de prova para cada tipo de liga testada, que foram trituradas mecanicamente, dando um total de 200 espécimes e, posteriormente armazenados a 37ºC ñ 1ºC, nas condiçöes de 1 hora, 24 horas, 7 dias e 14 dias. Após a armazenagem media-se o comprimento do corpo de prova que, em seguida, era imerso em uma soluçäo de 5ml de ácido cítrico a 5 por cento e 1 ml de ácido fosfomolíbdico a 1 por cento. Decorridos 10 minutos, sob agitaçäo mecânica, o líquido foi tarnsferido para um tubo de ensaio e levado ao espectrofotômetro, regulado em 578 namômetros, para a leitura da densidade óptica...

Nickel (II)-induced apoptosis and G2/M enrichment

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 microM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 microM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 microM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.

Chromosome sensitivity to bleomycin in G2 lymphocytes from Down syndrome patients

Inúmeros trabalhos têm demonstrado que linfócitos de pacientes com síndrome de Down apresentam uma maior freqüência de aberraçöes cromossômicas quando expostos a radiaçäo ionizante ou agentes químicos nas fases G0 ou G1 do ciclo celular, mas näo em G2, quando comparados com controles normais. Para determinar a sensibilidade de linfócitos de pacientes com síndrome de Down, na fase G2, usou-se o radiomimético bleomicina em culturas de linfócitos de 24 pacientes. Todos os pacientes mostraram trissomia livre do cromossomo 21 (47,XX + 21 ou 47, XY + 21). Indivíduos que apresentaram freqüência média de quebras cromatídicas por célula superior a 0,8 foram considerados sensíveis a droga. Nenhum controle apresentou suscetibilidade a bleomicina e entre os 24 pacientes com síndrome de Down somente um foi sensível à droga. Näo se observou qualquer diferença significativa entre os dois grupos em relaçäo as freqüências de quebras cromatídicas em linfócitos em G2, o que está de acordo com outros trabalhos. A distribuiçäo das quebras induzidas pela bleomicina, em cada grupo cromossômico, foi igual para pacientes e controles. Nenhuma diferença significativa foi observada na resposta a bleomicina entre homens e mulheres, nos dois grupos. Provavelmente, o principal fator envolvido na sensibilidade cromossômica de linfócitos de pacientes com síndrome de Down seja a fase do ciclo celular na qual a célula é tratada.

Differential effect of cordycepin on S and G2 phases of cell cycle in plasmodia of Physarum polycephalum Schw.

Effect of pulse treatments of cordycepin, an analog of adenosine, on S and G2 phases of the cell cycle of the mitotically synchronous plasmodia of Physarum polycephalum has been studied. Various concentrations of the drug (50-200 micrograms ml-1) were found to be effective in delaying mitosis by several hours in both the phases. However, there was a significant increase in mitotic delay in those treated during G2. It is suggested that this extra delay during G2 could be due to the transcriptive level inhibition of specific RNA types, such as that of tubulins, whose gene activity is cell cycle regulated and turned on during G2 in Physarum, or alternatively because of a deficiency for ATP and the consequent inhibition of events such as mitotic spindle assembly and phosphorylation of histones.