Phase Arrest In HeLa Cells Research Articles

DNA binding, cleavage and anticancer activity of a Zn(II)-Cu(II) hetero-dinuclear carbohydrazone complex containing the secondary ligand 1,10-phenanthroline

A hetero di-nuclear complex [Zn(o-phen)LCu(OAc)] (1) (H3L = o-HOC6H4CH = NNH)2C = O, o-phen = 1,10-phenanthroline) exhibited intercalative DNA binding (Kb = 1.03 × 105 M−1) as revealed from UV–Vis spectral analysis as well as viscosity study and further supported by molecular docking study of 1 with DNA. The apparentbinding constant (Kapp = 21.78 × 105 M−1) obtained from ethidium bromide (EB) displacement assay using fluorescence spectral study further confirmed the efficient binding ability of the complex to DNA. The complex displayed both oxidative as well as hydrolytic DNA cleavage activity. Quantitative hydrolytic cleavage results suggested significant nuclease activity against PUC19 plasmid DNA. Oxidative DNA cleavage experiments done with ROS (reactive oxygen species) scavengers showed the involvement of •OH in the cleavage mechanism. Anticancer activity of 1 was tested by evaluating the cytotoxicity of the complex towards cervical cancer HeLa, cisplatin resistant breast cancer MDA-MB-231 and epidermoid carcinoma A431 cells. IC50 values (11.08 ± 1.1 µM for HeLa, 2.75 ± 0.16 µM for MDA-MB-231 and 1.38 ± 0.07 µM for A431 cells) revealed significant cell death in all three cell lines. Nuclear staining using AO/PI and Hoechst 33,342 suggested cell death occurred due to apoptosis in these cancer cells. 1 induced high level of ROS in HeLa cells. It also caused G2/M phase arrest in HeLa cells as revealed from cell cycle analysis.

Rapid DNA interstrand cross-linking of Pt(IV) compound

Pt(IV) anticancer compounds have been developed for several decades to overcome the drawbacks of their Pt(II) congeners, and the reduction of Pt(IV) to Pt(II) has been commonly regarded as a necessary step in the activation of Pt(IV) compounds prior to targeting DNA. However, blockage of glutathione (GSH) biosynthesis resulted in a slight effect on the cytotoxicity of oxoplatin in yeast Saccharomyces cerevisiae strains, urging us to reconsider the mechanism of actions for the “inert” Pt(IV) complexes. Using X-ray absorption near-edge spectroscopy (XANES), our data demonstrated that Pt(IV) complex oxoplatin could bind to DNA in a tetravalent state. Both alkaline denaturing agarose electrophoresis and thermal denaturation-renaturation assay revealed that oxoplatin could rapidly produce stable interstrand crosslinks (ICLs), which can further translate into a fast cell-killing process in cancer cells. Using quantitative real-time PCR and immunofluorescence analysis, we also proved that Pt(IV) complex oxoplatin could induce a quick intracellular response of the FA/BRCA pathway in cancer cells that involves the DNA interstrand crosslinking repair system, and this quick response to ICLs was independent with the intracellular GSH levels. Cell cycle analysis showed that short incubation with oxoplatin can induce a strong S phase arrest in HeLa cells, indicating that the rapid interstrand crosslinks produced by oxoplatin might stall the replication fork, result in the double-strand breaks, and eventually induce cell death. Our results implied that, besides the reduction mechanism to release the Pt(II) congeners, direct and rapid interstrand cross-linking with DNA by Pt(IV) compounds might be a unique mechanism for Pt(IV) compounds, which may provide new insight for the development of next-generation platinum-based drugs.

Synthesis, biological evaluation, and structure-activity relationships of new tubulin polymerization inhibitors based on 5-amino-1,2,4-triazole scaffold

Based on our previous research, thirty new 5-amino-1H-1,2,4-triazoles possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative activities. Among them, compounds IIa, IIIh, and IIIm demonstrated significant antiproliferative activities against a panel of tumor cell lines, and the promising compound IIIm dose-dependently caused G2/M phase arrest in HeLa cells. Furthermore, analogue IIa exhibited the most potent tubulinpolymerization inhibitory activity with an IC50 value of 9.4 μM, and molecular modeling studies revealed that IIa formed stable interactions in the colchicine-binding site of tubulin, suggesting that 5-amino-1H-1,2,4-triazole scaffold has potential for further investigation to develop novel tubulin polymerization inhibitors with anticancer activity.

Induction of apoptotic effects of anti-proliferative zeolite X from coal fly ash on cervical cancer (HeLa) cell lines.

The synthesised zeolite X from coal fly ash showed significant cytotoxic activity in contradiction of HeLa cells (cervical cancer) in a concentration-dependent way at concentrations ranges from 200µg to 0.781µg/ml as shown by MTT assay and failed to cause cytotoxic effect in normal cells (Gh239). Cell cycle analysis exposed that zeolite X (10 and 15µg/ml) endorses cell growth inhibition by inducing G2/M phase arrest in HeLa cells as observed using flow cytometry. The confocal microscopic results depicted increased early apoptotic related changes in HeLa cell lines induced by zeolite X at a dosage of 10, 15 and 20µg/ml. Zeolite X at a dosage of 10, 15 and 20µg/ml in HeLa cells showed fragmentation of DNA by ladder pattern thereby indicates that cell death is related with apoptosis. By the increase of Bax/Bcl-2 ratio, zeolite X leads to the caspase-3 and caspase-9 activation and allow the cells to enter apoptosis. These collective results evidently showed that the influence of mitochondria-mediated signalling pathway in zeolite X induced apoptosis and intensely delivered investigational suggestion for the use of zeolite X as a significant curative agent in the preclusion and therapy of human cervical carcinoma.

SH003‑induced G1 phase cell cycle arrest induces apoptosis in HeLa cervical cancer cells.

Cervical cancer is a prevalent disease that may lead to mortality in women. In spite of the development of common therapeutic agents to treat cancer, there are several limitations of their use owing to side effects and drug resistance, which may induce cancer recurrence. The anticancer effects of the new herbal mixture SH003 (comprising Astragalusmembranaceus, Angelicagigas and Trichosantheskirilowii Maximowicz) have been examined in various types of cancer. Thus, the present study hypothesized that SH003 may be an effective treatment for cervical cancer. SH003 treatment inhibited the growth of HeLa cells, whereas it did not affect the growth of rat intestinal epithelial cells. In addition, SH003 treatment increased the expression of apoptosis‑related proteins and promoted apoptotic cell death in HeLa cells. SH003 treatment also led to G1 phase arrest in HeLa cells. Furthermore, SH003 treatment induced the production of reactive oxygen species (ROS); however, ROS production did not appear to be related to SH003‑mediated apoptosis. Results from the present study indicated that the SH003‑induced inhibition of HeLa cell growth may be mediated through G1 phase arrest and extrinsic apoptosis, suggested that SH003 may be a potential treatment for cervical cancer.

Baicalein inhibits TNF-α-induced NF-κB activation and expression of NF-κB-regulated target gene products.

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, apoptosis and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified baicalein from Scutellaria baicalensis as an inhibitor of NF-κB activation. As examined by the NF-κB luciferase reporter assay, we found that baicalein suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. It also inhibited TNF-α-induced nuclear translocation of p65 through inhibition of phosphorylation and degradation of IκBα. Furthermore, baicalein blocked the TNF-α-induced expression of NF-κB target genes involved in anti-apoptosis (cIAP-1, cIAP-2, FLIP and BCL-2), proliferation (COX-2, cyclinD1 and c-Myc), invasion (MMP‑9), angiogenesis (VEGF) and major inflammatory cytokines (IL-8 and MCP1). The flow cytometric analysis indicated that baicalein potentiated TNF-α-induced apoptosis and induced G1 phase arrest in HeLa cells. Moreover, baicalein significantly blocked activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2). Our results imply that baicalein could be a lead compound for the modulation of inflammatory diseases as well as certain cancers in which inhibition of NF-κB activity may be desirable.

Improvement in antiproliferative activity of Angelica gigas Nakai by solid dispersion formation via hot-melt extrusion and induction of cell cycle arrest and apoptosis in HeLa cells.

Angelica gigas Nakai (AGN) is one of the most popular herbal medicines and widely used as a functional food product. In this study, AGN was firstly processed by a low-temperature turbo mill and a hot melting extruder to reduce particle size and form solid dispersion (SD). Anticancer activity against HeLa cells was then examined. AGN-SD based on Soluplus was formed via hot-melt extrusion (HME) and showed the strongest cytotoxic effect on HeLa cells. In addition, the possible mechanism of cell death induced by AGN-SD on HeLa cells was also investigated. AGN-SD decreased cell viability, induced apoptosis, increased the production of reactive oxygen species, regulated the expression of Bcl-2 and Bax, and induced G2/M phase arrest in HeLa cells. This study suggested that AGN-SD based on Soluplus and the method to improve antiproliferative effect by SD formation via HME may be suitable for application in the pharmaceutical industry.

Polypeptide Fraction fromArca subcrenataInduces Apoptosis and G2/M Phase Arrest in HeLa Cells via ROS-Mediated MAPKs Pathways

Arca subcrenata is documented in the literature of marine Traditional Chinese Medicine. Polypeptide fraction from A. subcrenata, coded as P2, was demonstrated to possess significant antitumor activity in our previous study. However, the underlying mechanism remains undefined. The present study was carried out to investigate the underlying antitumor mechanism of P2 in human cervical cancer HeLa cells by MTT, FCM, LSCM, and western blot assays. The results revealed that P2 significantly induced apoptosis of HeLa cells in a concentration- and time-dependent manner. High level of ROS was provoked by P2, which was in turn responsible for induction of apoptosis through activation of intrinsic mitochondrial pathway and JNK1/2, p38 MAPK pathways, as well as inhibition of ERK1/2 pathway, as evidenced by the abrogation of P2's effect on HeLa cells preincubated with the ROS scavenger NAC. P2 also was observed to display significant effect on G2/M phase arrest by downregulating the expression of cyclin B1/cdc2 complex and upregulating the expression of p21. These findings demonstrate that P2 induces apoptosis and G2/M phase arrest in HeLa cells through ROS-mediated MAPKs pathways, suggesting that P2 would be worth investigating as a promising agent within the scope of marine drugs for treatment of cervical cancer.

Anticancer properties of extracts from Opuntia humifusa against human cervical carcinoma cells.

In this study, we found that the total polyphenol and ascorbic acid levels in the fruit of Opuntia humifusa are higher than those in other parts of the plant. We further hypothesized that antioxidants in O. humifusa might affect the growth or survival of cancer cells. Hexane extracts of seeds and ethyl acetate extracts of fruits and stems significantly suppressed the proliferation of HeLa cervical carcinoma cells, but did not affect the proliferation of normal human BJ fibroblasts. Additionally, the extracts of O. humifusa induced G1 phase arrest in HeLa cells. The O. humifusa extracts reduced the levels of G1 phase-associated cyclin D1, cyclin-dependent kinase 4 (Cdk4), and phosphorylated retinoblastoma proteins. Moreover, p21(WAF1/Cip1) and p53 expression significantly increased after treatment. We examined the effects of ethyl acetate extracts of O. humifusa fruit (OHF) on HeLa cells xenograft tumor growth. OHF treatment significantly reduced tumor volume and this decrease was correlated with decreased Cdk4 and cyclin D1 expression. Furthermore, flavonoids, trans Taxifolin, and dihydrokaempferol, were isolated from OHF. Thus, this extract may be a promising candidate for treating human cervical carcinoma.

Aurora-A kinase-inactive mutants disrupt the interaction with Ajuba and cause defects in mitotic spindle formation and G2/M phase arrest in HeLa cells

Aurora-A is a centrosome-localized serine/threonine kinase that is overexpressed in multiple human cancers. We previously reported an intramolecular inhibitory regulation of Aurora-A between its N-terminal regulatory domain (Nt, amino acids [aa] 1-128) and the C-terminal catalytic domain (Cd, aa 129-403). Here, we demonstrate that although both Aurora-A mutants (AurA-K250G and AurA-D294G/Y295G) lacked interactions between the Nt and Cd, they also failed to interact with Ajuba, an essential activator of Aurora-A, leading to loss of kinase activity. Additionally, overexpression of either of the mutants resulted in centrosome amplification and mitotic spindle formation defects. Both mutants were also able to cause G2/M arrest and apoptosis. These results indicate that both K250 and D294/Y295 are critical for direct interaction between Aurora-A and Ajuba and the function of the Aurora-A complex in cell cycle progression. [BMB Reports 2014; 47(11): 631-636]

Novel microtubule-targeted agent 6-chloro-4-(methoxyphenyl) coumarin induces G2-M arrest and apoptosis in HeLa cells

To identify a novel coumarin analogue with the highest anticancer activity and to further investigate its anticancer mechanisms. The viability of cancer cells was investigated using the MTT assay. The cell cycle progression was evaluated using both flow cytometric and Western blotting analysis. Microtubule depolymerization was observed with immunocytochemistry in vivo and a tubulin depolymerization assay in vitro. Apoptosis was demonstrated using Annexin V/Propidium Iodide (PI) double-staining and sub-G(1) analysis. Among 36 analogues of coumarin, 6-chloro-4-(methoxyphenyl) coumarin showed the best anticancer activity (IC(50) value about 200 nmol/L) in HCT116 cells. The compound had a broad spectrum of anticancer activity against 9 cancer cell lines derived from colon cancer, breast cancer, liver cancer, cervical cancer, leukemia, epidermoid cancer with IC(50) value of 75 nmol/L-1.57 μmol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC(50) value of 12.128 μmol/L). The compound (0.04-10 μmol/L) induced G(2)-M phase arrest in HeLa cells in a dose-dependent manner, which was reversible after the compound was removed. The compound (10-300 μmol/L) induced the depolymerization of purified porcine tubulin in vitro. Finally, the compound (0.04-2.5 μmol/L) induced apoptosis of HeLa cells in dose- and time-dependent manners. 6-Chloro-4-(methoxyphenyl) coumarin is a novel microtubule-targeting agent that induces G(2)-M arrest and apoptosis in HeLa cells.

Osmium(vi) nitrido complexes bearing azole heterocycles: a new class of antitumor agents

Eight new nitridoosmium(VI) complexes with the general formula [OsVI(N)Cl3(Hazole)2] have been synthesized and characterized. The cellular uptake and the antiproliferative activities of these compounds against a panel of human cancer cell lines have been investigated. Complexes with pyrazole derivatives (1, 2, 3 and 4) are found to possess significant in vitro cytotoxicity. Further studies of compounds 1 and 3 indicate that they induce S phase arrest in HeLa cells followed by apoptosis, possibly as a result of binding with DNA.

Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells

Wogonin, a naturally occurring flavonoid, has been shown to have tumor therapeutic potential both in vitro and in vivo. To better understand its anticancer mechanism, we examined the effect of wogonin on human cervical carcinoma HeLa cells. In this study, we observed that G1 phase arrest was involved in wogonin-induced growth inhibition in HeLa cells. Over a 24 h exposure of HeLa cells to 90 micromol x L(-1) wogonin, the promoters of G1-S transition, including cyclin D1/Cdk4 and pRb, decreased within 12 h and E2F-1 depleted in the nucleus at the same time. As the G1 phase arrest developed, p53 and the Cdk inhibitor p21Cip1 elevated both at protein and mRNA levels. Furthermore, the up-regulation of p21Cip1 induced by wogonin was dramatically inhibited by siRNA-mediated p53 gene silencing. Collectively, our data suggested that wogonin induced G1 phase arrest in HeLa cells by modulating several key G1 regulatory proteins, such as Cdk4 and cyclin D1, as well as up-regulation of a p53-mediated p21Cip1 expression. This mechanism of wogonin may play an important role in the killing of cancerous cells and offer a potential mechanism for its anticancer action in vivo.

HY251, a novel cell cycle inhibitor isolated from Aralia continentalis, induces G 1 phase arrest via p53-dependent pathway in HeLa cells

This study was aimed to elucidate the novel structure of HY251 isolated from the roots of Aralia continentalis and to evaluate its detailed inhibition mechanisms on cell cycle progression in HeLa cells. The structure of HY251 was elucidated based on the interpretation of the NMR spectra, as 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[ a]indene-3,3a,7a,8a-tetraol. The flow cytometric analysis revealed an appreciable G 1 phase arrest in HeLa cells treated with 100 μM of HY251. This HY251-induced G 1 phase arrest is associated with decreased expression of cyclin D3 and up-regulation of p21 CIP1 and p27 KIP1, via p53 phosphorylation at Ser-15 by transcriptional up-regulation of ATM, which resulted in increased hypophosphorylated pRb in HeLa cells.

Transient Failure to Dephosphorylate the cdc2-Cyclin B1 Complex Accompanies Radiation-Induced G 2 -Phase Arrest in HeLa Cells

Ionizing radiation causes a division delay in mammalian cells, dominated by a period of G2-phase arrest. The G2- to M-phase transition in dividing mammalian cells is dependent on the kinase activity of the cdc2-cyclin B protein complex. In the present investigation we measured the quantities of these two proteins, the formation of their complex and the kinase activity of the complex as a function of cell age in the cell cycle for irradiated and control mammalian cell populations. The human HeLa S3 cells were synchronized at the G1/S-phase border by double thymidine block and exposed 3 h after release to 1.75 Gy of X rays. Studies of HeLa cells at other laboratories have shown that, for doses of 5 Gy or more, division delay is associated with a suppression of production of cyclin B mRNA. Here we report that, for cells irradiated with low doses, there is a transient failure of the complex to activate which correlates with the duration of radiation-induced G2-phase arrest. The irradiated cells showed an increase in both cyclin B and phosphorylated cdc2 over the levels in control cells, and both persisted for a much longer period than in controls, further confirmation of delay in the activation of the catalytic subunit.