Cytotoxic activity and G2/M cell cycle arrest mediated by antofine,a phenanthroindolizidine alkaloid isolated from Cynanchum paniculatum

Starting with an extract derived from the root of Cynanchum paniculatum Kitagawa (Asclepiadaceae) that was active in the process of inhibiting the growth of human cancer cells in culture, a phenanthroindolizidine alkaloid antofine was isolated and identified as an active principle (IC50 = 7.0 +/- 0.2 ng/ml for A549, human lung cancer cells; IC50 = 8.6 +/- 0.3 ng/ml for Col2, human colon cancer cells). Prompted by the high potency of cancer cell growth inhibition, additional action mechanism studies were performed with antofine. Utilizing cultured Col2 cells as a model, antofine induced arrest in the G2/M phase of the cell cycle after 48 h of incubation. With wash-out experiment, colony formation was also inhibited in a dose-dependent manner. These data suggest the potential of antofine to serve as a cancer chemotherapeutic agent by virtue of arresting the cell cycle.     

Difference of cell cycle arrests induced by lidamycin in human breast cancer cells

Lidamycin (LDM) is a member of the enediyne antibiotic family. It is undergoing phase I clinical trials in China as a potential chemotherapeutic agent. In the present study, we investigated the mechanism by which LDM induced cell cycle arrest in human breast cancer cells. The results showed that LDM induced G1 arrest in p53 wild-type MCF-7 cells at low concentrations, and caused both G1 and G2/M arrests at higher concentrations. In contrast, LDM induced only G2/M arrest in p53-mutant MCF-7/DOX cells. Western blotting analysis indicated that LDM-induced G1 and G2/M arrests in MCF-7 cells were associated with an increase of p53 and p21, and a decrease of phosphorylated retinoblastoma tumor suppressor protein, cyclin-dependent kinase (Cdk), Cdc2 and cyclin B1 protein levels. However, LDM-induced G2/M arrest in MCF-7/DOX cells was correlated with the reduction of cyclin B1 expression. Further study indicated that the downregulation of cyclin B1 by LDM in MCF-7 cells was associated with decreasing cyclin B1 mRNA levels and promoting protein degradation, whereas it was only due to inducing cyclin B1 protein degradation in MCF-7/DOX cells. In addition, activation of checkpoint kinases Chk1 or Chk2 maybe contributed to LDM-induced cell cycle arrest. Taken together, we provide the first evidence that LDM induces different cell cycle arrests in human breast cancer cells, which are dependent on drug concentration and p53 status. These findings are helpful in understanding the molecular anti-cancer mechanisms of LDM and support its clinical trials.     

Flow cytometric evaluation of the cell cycle perturbations induced by S12363, a new vinca alkaloid derivative

L1210 cells treated for 21 hours with S12363, a new vinca alkaloid derivative and the parent compounds (vinblastine, vincristine, vindesine) at equitoxic concentrations were found, by flow cytometry, to be equally accumulated in the G2 + M phase of the cell cycle. The chromatin structure of these cells was then analyzed in order to quantify with high precision the percentage of cells in mitosis. S12363 was found to accumulate, from the first hours of treatment (4-8 hours), and at lower concentrations, a higher percentage of cells in the M phase than the reference drugs. Taking into account previously published studies concerning the characteristics of vinblastine and vincristine uptake, our results are compatible with a facilitated uptake of S12363.     

Cimiside E arrests cell cycle and induces cell apoptosis in gastric cancer cells

Cimiside E was isolated from the Cimicifuga heracleifolia Komarov extract, which has been previously demonstrated to possess apoptotic action on gastric cancer cells. The IC₅₀ value of cimiside E on gastric cancer cells for 24 h was 14.58 μM. The mechanism of apoptosis was further elucidated through western blot, RT-PCR, morphology, Annexin V-FITC/PI staining and cell cycle analysis. Cell cycle arrest was induced by cimiside E in S phase at a lower concentration (30 μM) and G2/M phase at higher concentrations (60 and 90 μM). Cimiside E mediated apoptosis through the induction of the caspase cascade for both the extrinsic and intrinsic pathways. These findings suggest that cimiside E may be an effective chemopreventive agent against cancer.     

Tubulozole-induced G2/M cell cycle arrest in human colon cancer cells through formation of microtubule polymerization mediated by ERK1/2 and Chk1 kinase activation.

Our studies demonstrated that human colon cancer cells (COLO 205), with higher expression level of check point kinase 1 (Chk1), were more sensitive to microtubule damage agent Tubulozole (TUBU) induced G2/M phase arrest than normal human colon epithelial (CRL) cells. TUBU (10 microM, for 3h) treatment resulted in rapid and sustained phosphorylation of Cdc25C (Ser-216) leading to increased 14-3-3beta binding. This resulted in increased nuclear translocation. In addition, TUBU induced phosphorylation of the Cdc25C (Ser-216) and Bad (Ser-155) proteins were blocked by Chk1 SiRNA-transfection. Surprisingly, cellular apotosis was observed in cells treated with TUBU after Chk1 SiRNA inhibition. We further demonstrated that extracellular signal-regulated kinase (ERK) activation by TUBU was needed for Chk1 kinase activation and microtubule formation as shown by the attenuation of these responses by the ERK1/2 specific inhibitor PD98059. However, TUBU induced ERK1/2 phosphorylation was not blocked in the Chk1 SiRNA-transfected COLO 205 cells. These results imply that ERK1/2 mediated Chk1 activation may be play an important role in determining TUBU induced G2/M arrest or apoptosis in COLO 205 cells.     

A new diaryl urea compound, D181, induces cell cycle arrest in the G1 and M phases by targeting receptor tyrosine kinases and the microtubule skeleton

Receptor tyrosine kinases (RTKs) modulate a variety of cellular events, including cell proliferation, differentiation, mobility and apoptosis. In addition, RTKs have been validated as targets for cancer therapies. Microtubules are another class of proven targets for many clinical anticancer drugs. Here, we report that 1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-cyano-4-hydroxyphenyl)urea (D181) functions as both a receptor tyrosine kinase inhibitor and a tubulin polymerization enhancer. D181 displayed potent inhibitory activities against a panel of RTKs, including Flt3, VEGFR, cKit, FGFR1 and PDGFRβ. D181 also enhanced tubulin polymerization and modified the secondary structure of tubulin proteins to disrupt their dynamic instability. Because of synergistic cooperation, D181 strongly inhibited the proliferation of various cancer cell lines, induced LoVo cell cycle arrest in the G1 and M phases and suppressed tumor growth in nude mice bearing human LoVo and HT29 xenografts. Our studies have provided a new, promising lead compound and novel clues for multi-target anticancer drug design and development.     

17Alpha-estradiol arrests cell cycle progression at G2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

A pharmacological dose (2.5-10 μM) of 17α-estradiol (17α-E₂) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17α-E₂ was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G₂/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17α-E₂-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G₂/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17α-E₂-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G₁/S boundary, 17α-E₂ failed to induce the G₂/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17α-E₂ toward Jurkat T cells is attributable to apoptosis mainly induced in G₂/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.     

Suppression of cell cycle progression by a fungal lectin: activation of cyclin-dependent kinase inhibitors

The antiproliferative activity of a fungal lectin (VVL) isolated from the mushroom, Volvariella volvacea, was studied using a battery of cultured tumor cell lines. It was revealed that [(3)H]thymidine incorporation into the cell lines was markedly reduced at 0.32 microM VVL. When S180 mouse sarcoma cells were incubated for 48 hr with doses of VVL ranging from 0.32 to 0.8 microM, prominent blebs on the cell surface and large vacuoles in the cytoplasm, but not apoptotic bodies, were observed under a fluorescence microscopy. VVL did not exert ribosome-inactivating activity or induce any changes in the expression of cyclins A, D1, and E. However, it did activate the expression of cyclin kinase inhibitors, namely p21, p27, p53, and Rb, in a dose-dependent manner. Flow cytometric analysis demonstrated an accumulation of cells in the G2/M phase in a time- and dose-dependent manner, indicating that VVL arrested cell proliferation by blocking cell cycle progression in the G2/M phase.     

MT7, a novel compound from a combinatorial library,arrests mitosis via inhibiting the polymerization of microtubules

Targeting cellular mitosis is an attractive antitumor strategy. Here, we reported MT7, a novel compound from the 6H-Pyrido[2′,1′:2,3]imidazo [4,5-c]isoquinolin- 5(6H)-one library generated by using the multi-component reaction strategy, as a new mitotic inhibitor. MT7 elicited apparent inhibition of cell proliferation by arresting mitosis specifically and reversibly in various tumor cell lines originating from different human tissues. Detailed mechanistic studies revealed that MT7 induced typical gene expression profiles related to mitotic arrest shown by cDNA microarray assays. Connectivity Map was used to analyze the microarray data and suggested that MT7 was possibly a tubulin inhibitor due to its similar gene expression profiles to those of the known tubulin inhibitors demecolcine, celastrol and paclitaxel. Further analyses demonstrated that MT7 inhibited the polymerization of cellular microtubules although it was not detectable to bind to purified tubulin. The inhibition of cellular tubulin polymerization by MT7 subsequently resulted in the disruption of mitotic spindle formation, activated the spindle assembly checkpoint and consequently arrested the cells at mitosis. The persistent mitotic arrest by the treatment with MT7 led the tested tumor cells to apoptosis. Our data indicate that MT7 could act as a promising lead for further optimization, in hopes of developing new anticancer therapeutics and being used to probe the biology of mitosis, specifically, the mode of interference with microtubules.     

Growth inhibition and induction of G(1) phase cell cycle arrest in neuroblastoma SH-SY5Y cell by tri-ortho-cresyl phosphate

It has been known that tri-ortho-cresyl phosphate (TOCP) can induce delayed neurotoxicity in humans and sensitive animal species; however, it also has influence on the developing central nervous system or differentiating neuronal cells. In this study, the effects of TOCP on cell proliferation and cell cycle regulation and the mechanisms that contribute to this effect were investigated by using human neuroblastoma SH-SY5Y cell line. Treatment of the cells with TOCP suppressed cell proliferation and reduced cell viability in a dose- and time-dependent manner. Analysis of cell cycle profile indicated that TOCP blocked cell cycle progression by arresting the cell cycle at G(1) phase. The data of determination of cell cycle regulated molecules at mRNA and protein levels showed that TOCP decreased cyclin D1 and increased p21 expression, while did not affect the p53 and p27 levels. Thus, these results indicated that TOCP might induce potential neurodevelopmental toxicity, and a possible mechanism of this toxicity might be the disturbance of cell proliferation by disrupting cell cycle regulatory proteins cyclin D1 and p21 expression.     

Inhibition of microtubule polymerization by SK&F 96365, a blocker of receptor-linked Ca2+ entry

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imid azole hydrochloride) is widely used as an effective inhibitor of receptor-linked and capacitative Ca2+ entry. Since this inhibitor has additional effects such as inhibition of voltage-dependent Ca2+ channels, sarco- and endoplasmic reticula Ca2+ pumps and cell proliferation, its molecular mechanism of action remains to be solved. In the present study, we have investigated the effect of SK&F 96365 on microtubule protein isolated from bovine brain in vitro. SK&F 96365 depolymerized the polymerized microtubules in a concentration-dependent manner. This result suggests that SK&F 96365 directly depolymerizes microtubules, an effect that may contribute to the various actions of this compound.     

新型微管抑制剂Yf9b能够诱导Hela细胞G2/M期阻滞和凋亡

目的考察新型微管抑制剂2-甲氧基-5-(4-(3,4,5-三家氧基苯基)-1,3-硒唑-5-基)苯胺[2-methoxy-5-(4-(3,4,5-trimethoxyphenyl)-1,3-selenazol-5-yl)aniline,Yf9b]对多种人源性肿瘤细胞的抗肿瘤活性,并研究Yf9b抑制人宫颈癌Hela细胞增殖的机制。方法采用MTT法考察Yf9b对多种肿瘤细胞的增殖抑制作用。免疫荧光染色观察Yf9b与康普瑞丁(combretastatin A-4,CA-4)对微管的抑制作用。流式细胞技术考察Yf9b与CA-4对Hela细胞周期的影响,并考察Yf9b诱导Hela细胞凋亡的情况。结果 Yf9b的作用时间与剂量依赖性地抑制Hela细胞增殖,半数抑制浓度(IC50)为(42.9±2.7)nmol·L-1。Yf9b同母体化合物CA-4一样能抑制微管聚合,将Hela细胞阻滞在G2/M期,并最终诱导细胞凋亡。结论 Yf9b是一种新型微管抑制剂,能够诱导Hela细胞G2/M期阻滞和凋亡。     

Taxanes: microtubule and centrosome targets,and cell cycle dependent mechanisms of action

Microtubules are highly dynamic cellular polymers made of alphabeta-tubulin and associated proteins. They play a key role during mitosis, participating in the exact organization and function of the spindle, and are critical for assuring the integrity of the segregated DNA. Therefore, they represent one of the more effective targets in current cancer therapy. Paclitaxel (Taxol) is the prototype of the taxane family of antitumor drugs, and it was the first natural product shown to stabilize microtubules. This unique mechanism of action is in contrast to other microtubule poisons, such as Vinca alkaloids, colchicine, and cryptophycines, which inhibit tubulin polymerization. Taxanes block cell cycle progression through centrosomal impairment, induction of abnormal spindles and suppression of spindle microtubule dynamics. Triggering of apoptosis by aberrant mitosis or by subsequent multinucleated G1-like state related to mitotic slippage, depends on cell type and drug schedule. The development of fluorescent derivatives of paclitaxel led us to locate spindle pole microtubules and centrosomes as main sub-cellular targets of cytotoxic taxoids in living cells. In this review we discuss these findings in the context of a cell cycle-dependent response to taxanes, based on the cellular targets, and the status of the implicated cell cycle checkpoints. We also review those events that can influence this response, like the different signal transduction pathways activated/inactivated in relation to Bcl-2 phosphorylation and induction of apoptosis, and the controversial role of the p53 status on cell sensitivity to paclitaxel. Finally, cell cycle-dependent resistance, an emerging concept in combination sequential chemotherapy, is discussed on the basis of the cell cycle-dependent mechanisms of action of taxanes.     

Pirarubicin inhibits multidrug-resistant osteosarcoma cell proliferation through induction of G2/M phase cell cycle arrest

Aim:

Pirarubicin (THP) is recently found to be effective in treating patients with advanced, relapsed or recurrent high-grade osteosarcoma. In this study, the effects of THP on the multidrug-resistant (MDR) osteosarcoma cells were assessed, and the underlying mechanisms for the disruption of cell cycle kinetics by THP were explored.

Methods:

Human osteosarcoma cell line MG63 and human MDR osteosarcoma cell line MG63/DOX were tested. The cytotoxicity of drugs was examined using a cell proliferation assay with the Cell Counting Kit-8 (CCK-8). The distribution of cells across the cell cycle was determined with flow cytometry. The expression of cell cycle-regulated genes cyclin B1 and Cdc2 (CDK1), and the phosphorylated Cdc2 and Cdc25C was examined using Western blot analyses.

Results:

MG63/DOX cells were highly resistant to doxorubicin (ADM) and gemcitabine (GEM), but were sensitive or lowly resistant to THP, methotrexate (MTX) and cisplatin (DDP). Treatment of MG63/DOX cells with THP (200–1000 ng/mL) inhibited the cell proliferation in time- and concentration-dependent manners. THP (50–500 ng/mL) induced MG63/DOX cell cycle arrest at the G₂/M phase in time- and concentration-dependent manners. Furthermore, the treatment of MG63/DOX cells with THP (200–1000 ng/mL) downregulated cyclin B1 expression, and decreased the phosphorylated Cdc2 at Thr¹⁶¹. Conversely, the treatment increased the phosphorylated Cdc2 at Thr¹⁴/Tyr¹⁵ and Cdc25C at Ser²¹⁶, which led to a decrease in Cdc2-cyclin B1 activity.

Conclusion:

The cytotoxicity of THP to MG63/DOX cells may be in part due to its ability to arrest cell cycle progression at the G₂/M phase, which supports the use of THP for managing patients with MDR osteosarcoma.     

Cadmium induces cell cycle arrest in rat kidney epithelial cells in G2/M phase

Cadmium (Cd) has been reported to cause cell cycle arrest in various cell types by p53-dependent and -independent mechanisms. This study was designed to investigate cell cycle progression in kidney cells that are the target of chronic Cd toxicity. Rat renal proximal tubular epithelial cells, NRK-52E, were treated with up to 20 microM CdCl2 in DMEM containing 10% calf serum for up to 24 h. Flow cytometric analysis revealed time- and concentration-dependent increases in cells in G2/M phase of the cell cycle. As compared to the control cells, the cells exposed to 20 microM Cd showed a doubling of the number of cells in this phase after 24 h. The cell cycle arrest was associated with a decrease in protein levels of both cyclins A and B. Further investigation into the mechanism revealed that Cd treatment led to down-modulation of cyclin-dependent kinases, Cdk1 and Cdk2, apparently by elevating the expression of cyclin kinase inhibitors, KIP1/p27 and WAF1/p21. Furthermore, the wild-type p53 DNA-binding activity was up-regulated. Based on these observations, it appears that Cd causes G2/M phase arrest in NRK-52E cells via elevation of p53 activity, increasing the expression of cyclin kinase inhibitors p27 and p21, and decreasing the expression of cyclin-dependent kinases Cdk1 and 2, and of cyclins A and B.     

(-)-Phenylahistin arrests cells in mitosis by inhibiting tubulin polymerization.

(-)-Phenylahistin, a fungal diketopiperazine metabolite composed of phenylalanine and isoprenylated dehydrohistidine, arrested cells in mitosis and inhibited the proliferation of A549 cells. The microtubule network in A549 cells was disrupted by (-)-phenylahistin, which also inhibited the polymerization of both microtubule protein from bovine brain and phosphocellulose-purified tubulin in vitro. Competitive binding studies indicated that (-)-phenylahistin interacted with the colchicine binding site on tubulin but not with the vinblastine binding site.     

Growth inhibition and induction of G1 phase cell cycle arrest in human lung cancer cells by a phenylbutenoid dimer isolated from Zingiber cassumunar

In our previous study, a novel phenylbutenoid dimer (+/-)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (PSC), isolated from Zingiber cassumunar ROXB. (Zingiberaceae), inhibited proliferation of various human cancer cells with the IC(50) values ranging 10 to 30 microM. Prompted by these anti-proliferative effects, we performed additional studies in A549 human lung cancer cells in order to investigate the mechanism of action. PSC arrested cell cycle progression at the G0/G1 phase in a concentration- and time-dependent manner. PSC dose-dependently induced cyclin-dependent kinase (CDK) inhibitor p21 expression, whereas the expression of cyclin D1, cyclin A, CDK4, CDK2, and proliferating cell nuclear antigen (PCNA) were decreased by treatment with PSC. These results suggest that one of the anti-proliferative mechanisms of PSC is to suppress cell cycle progression by increasing p21 expression and down-regulating cyclins and CDKs. This study characterizes additional biological activity of this novel phenylbutenoid dimer and expands its therapeutic potential for cancer as a chemotherapeutic agent derived from natural products.