The antiproliferative activity of saponin-enriched fraction from Bupleurum Kaoi is through Fas-dependent apoptotic pathway in human non-small cell lung cancer A549 cells

Bupleuri Radix (Chai-hu in Chinese and Saiko in Japanese) is one of the most important traditional Chinese crude drugs for treating hepatitis malaria and intermittent fever. B. kaoi is one of the Bupleurum spp. families locally found in Taiwan. The effects of saponin-enriched fraction (SEF) from Bupleurum Kaoi in human non-small cell lung cancer A549 cells were investigated in this study. An enhancement in Fas and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), was responsible for the apoptotic effect induced by SEF. Taken together, our study suggests that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of SEF in A549 cells.     

Antiproliferative activity of paeoniflorin is through cell cycle arrest and the Fas/Fas ligand-mediated apoptotic pathway in human non-small cell lung cancer A549 cells

1. Paeoniflorin (PF), isolated from the paeony root, is reported to have immunoregulatory, neuromuscular blocking, anticonvulsant, antihyperglycaemic and antihypotensive effects. 2. The present study investigated the antiproliferative activity of PF. The results showed that PF inhibited the proliferation of A549 by blocking cell cycle progression in the G(0)/G(1) phase and inducing apoptosis. 3. An ELISA showed that G(0)/G(1) phase arrest may be due to p53-independent induction of p21/wild-type p53-activated fragment 1 (WAF1). Increased protein expression of Fas/apoptosis-1 (APO-1) and its two ligands, membrane-bound Fas ligand and soluble Fas ligand, may be responsible for the PF-induced apoptosis. 4. This is the first study to show that the induction of p21/WAF1 and the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of PF in A549 cells.     

Antiproliferative activity of aucubin is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells

Aucubin, an iridoid glycoside isolated from the leaves of Aucuba japonica, inhibits human non-small cell lung cancer A549 cells by blocking cell cycle progression in the G(0)/G(1) phase and inducing apoptosis. An ELISA showed that the G(0)/G(1) phase arrest is due to p53-mediated induction of p21. Enhancement of Fas and its two ligands, membrane-bound and soluble Fas ligand, may be responsible for the apoptotic effect induced by aucubin. The present study shows, for the first time, that the induction of p53 and activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of aucubin in A549 cells.     

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra via Fas-mediated pathway

Myrica rubra Sieb et Zucc. (Myricaceae) is well known as a rich source of tannins. Prodelphinidin B-2 3,3'-di-O-gallate (PB233'OG) is a proanthocyanidin gallate that has been reported to exhibit antioxidant and antiviral activity. In this study, we evaluated the anti-proliferative activity of PB233'OG isolated from the bark of M. rubra in human breast adenocarcinoma MCF-7 cells. To identity the anti-cancer mechanism of PB233'OG, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that PB233'OG induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of PB233'OG-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of PB233'OG in MCF-7 cells.     

Prodelphinidin B-4 3'-O-gallate, a tea polyphenol, is involved in the inhibition of COX-2 and iNOS via the downregulation of TAK1-NF-kappaB pathway

Much is known about the bioactive properties of green tea flavan-3-ol. However, very little work has been done to determine the properties of proanthocyanidins, another kind of polyphenols in green tea. In this study, we have investigated the anti-inflammatory effect of tea prodelphinidin B-4 3'-O-gallate (PDG) by demonstrating the inhibitory effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. PDG caused a dose-dependent inhibition of COX-2 and iNOS at both mRNA and protein levels with the attendant decrease of prostaglandin E2 (PGE2) and nitric oxide (NO) production. Molecular data revealed that PDG downregulated NF-kappaB signaling pathway. Electrophoretic mobility shift assay (EMSA) showed that PDG reduced the binding complex of NF-kappaB-DNA in the promoter of COX-2 and iNOS. Immunochemical analysis revealed that PDG suppressed LPS-induced phosphorylation and degradation of IkappaBalpha, and subsequent nuclear translocation of p65. Consequently, PDG suppressed phosphorylation of IkappaB kinase alpha/beta (IKKalpha/beta) and TGF-beta-activated kinase (TAK1). Taken together, our data indicated that PDG is involved in the inhibition of COX-2 and iNOS via the downregulation of TAK1-NF-kappaB pathway, revealing partial molecular basis for the anti-inflammatory properties of tea PDG.     

Glabridin induces apoptosis and cell cycle arrest in oral cancer cells through the JNK1/2 signaling pathway

Glabridin, a flavonoid extracted from licorice (Glycyrrhiza glabra), possesses various biological properties, including anticancer activities. However, the effect of glabridin on oral cancer cell apoptosis and the underlying molecular mechanisms has not been elucidated. In this study, we demonstrated that glabridin treatment significantly inhibits cell proliferation in human oral cancer SCC‐9 and SAS cell lines. Flow cytometric assays demonstrated that glabridin induced several features of apoptosis, such as sub‐G1 phase cell increase and phosphatidylserine externalization. Furthermore, glabridin induced apoptosis dose‐dependently in SCC‐9 cells through caspase‐3, −8, and −9 activation and poly (ADP‐ribose) polymerase cleavage. Moreover, glabridin increased the phosphorylation of the extracellular signal–regulated kinase, p38, and c‐Jun N‐terminal kinase (JNK) pathways in a dose‐dependent manner. Moreover, the inhibition of the JNK1/2 inhibitor significantly reversed the glabridin‐induced activation of the caspase pathway. In conclusion, our findings suggest that glabridin induces oral cancer cell apoptosis through the JNK1/2 pathway and is a potential therapeutic agent for oral cancer.     

Novel combretastatin A-4 derivative XN0502 induces cell cycle arrest and apoptosis in A549 cells

Combretastatin A-4 (CA-4) is a tubulin-binding compound currently in phase II trial as a tumor vascular-targeting agent. The present study evaluates the anti-tumor activities and establishes the mechanism of the action of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine(XN0502), a novel synthesized CA-4 analogue, in an effort towards finding the favorable therapeutics of CA-4 derivatives. XN0502 is characterized by its more potent anti-proliferative activities against non-small cell lung cancer A549 cells (IC₅₀: 1.8 ± 0.6 μM), than that on the normal human liver HL-7702 cells (IC₅₀: 9.1 ± 0.4 μM). Of note, using tubulin polymerization assay, western blot and immuofluorescence analyses, XN0502 was showed to inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further studies indicated that XN0502 induced time- and dose-dependent G2/M arrest, accompanying with the reduction of CDC2/p34 expression and the downregulation of CDK7. The protein level alteration and the nuclear translocation of cyclinB1 were observed, denoting the M phase arrest in XN0502-treated cells. Moreover, XN0502 caused caspase-mediated apoptosis, as indicated by the cleavage of PARP, the reduction of procaspase-3 and procaspase-9, and the down-regulation of XIAP. Taken together, the current study demonstrates that the novel CA-4 analogue XN0502 is a promising anti-cancer agent with potent G2/M arrest- and apoptotic-inducing activities via targeting tubulin deserving further research and development, and helps provide data for exploiting new CA-4 analogues.     

Regulation of cell growth through cell cycle arrest and apoptosis in HPV 16 positive human cervical cancer cells by tea polyphenols

Cervical cancer is the second most common malignant neoplasm in women, in terms of both incidence and mortality rates worldwide. The polyphenolic constituents of tea (Camellia sinensis) have gained considerable attention because of its anti-cancer properties against a variety of cancers. Here we studied the effects of green and black tea polyphenols (GTP and BTP), on cellular proliferation and cell death in the SiHa cells (human cervical cancer) expressing the human papilloma virus (HPV)-16. The result showed that both GTP and BTP inhibited proliferation of cells in dose and time dependent manner. Cell cycle analysis showed anti-proliferative effect of GTP which is associated with an increase in the G2/M phase and apoptotic effect of BTP in 24 h treated SiHa cells. Further, on increase of incubation time for 48 h, GTP caused induction of apoptosis up to 20% of SiHa cells. The role GTP and BTP in apoptosis was further confirmed by reduction in mitochondrial membrane potential and increased levels of membrane phosphatidylserine. Thus, our data suggests that tea polyphenols exhibit anti-cancer potential against cervical cancer by inhibition of cell growth and induction of apoptosis.     

Mechanisms of induction of cell cycle arrest and cell death by cryptolepine in human lung adenocarcinoma a549 cells.

We investigated p53-dependent and -independent molecular events associated with cell cycle alteration and cell death in human lung adenocarcinoma A549 cells using cryptolepine, a DNA-damaging agent. After a 24-h treatment, cryptolepine caused an accumulation of p53 at concentrations of 1.25-10 microM and induction of p21(Cip1/WAF1) but only at concentrations up to 5muM. p21(Cip1/WAF1) was also strongly induced by cryptolepine (2.5-5 microM) in cells with p53 largely ablated via small interfering RNA-mediated gene silencing. Cryptolepine induced G1-phase block at 1.25-2.5 microM, S-phase and G2/M-phase block at 2.5-5 microM, and cell death at 10 microM. The dead cells displayed condensed and fragmented nuclei, features of apoptosis. Wortmannin, an inhibitor of ataxia telangiectasia-mutated and DNA-dependent protein kinase (DNA-PK), caused cell cycle arrest at G1 phase without inducing p53 and p21(Cip1/WAF1) expression and cell death. The addition of wortmannin partially prevented cryptolepine-induced expression of p53 and p21(Cip1/WAF1) together with the S-phase block and sensitized cells to induction of cell death. NU7026, a DNA-PK-specific inhibitor, showed neither induction of cell cycle arrest and apoptosis nor the expression of p53 and p21(Cip1/WAF1). The presence of NU7026 caused further reduction of cells in G1 phase induced by cryptolepine at 5 microM without affecting the induction of p53 and p21(Cip1/WAF1) and cell death. This study using the A549 cell as a model demonstrated that cryptolepine selects different molecular pathways to cell cycle checkpoint activation in a dose-specific manner and evokes a wortmannin-sensitive antiapoptosis response.     

Calycosin induces mitochondrial-dependent apoptosis and cell cycle arrest,and inhibits cell migration through a ROS-mediated signaling pathway in HepG2 hepatocellular carcinoma cells

Calycosin is one of the main ingredients extracted from the Chinese medical herb, Radix astragali (RA). It has been shown to inhibit cell proliferation and induce apoptosis in several cancer cell lines, but the underlying mechanism remains unclear. The effects of calycosin on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells, as well as its mechanism, were investigated in this study. Cell Counting Kit-8 assay results suggested that calycosin had anti-proliferation effects on HCC in dose- and time-dependent manners, and had less cytotoxicity in normal cells. Hoechst/PI double staining and flow cytometry results showed cellular morphological changes and apoptosis after treatment of HepG2 cells with calycosin. The western blot assay showed calycosin decreased the expression of Bcl-2 and increased the expression of Bax, caspase-3, and PARP. Calycosin induced the activation of MAPK, STAT3, NF-κB, apoptosis-related proteins, and induced cell cycle arrest in the G0/G1 phase by regulating AKT. In addition, calycosin reduced the expression of TGF-β1, SMAD2/3, SLUG, and vimentin. Furthermore, phosphorylation, apoptosis, and cell migration induced by calycosin were mediated by the production of reactive oxygen species. These events could be inhibited by pretreatment with N-acetyl-L-cysteine. Calycosin resisted HCC by activating ROS-mediated MAPK, STAT3, and NF-κB signaling pathways.     

Homocysteine induces cell cycle G1 arrest in endothelial cells through the PI3K/Akt/FOXO signaling pathway

OBJECTIVE: High levels of homocysteine (Hcy) induce a sustained injury on arterial endothelial cells, which accelerates the development of thrombosis and atherosclerosis. Hcy specifically inhibits the growth of endothelial cells. The present study investigated the signaling pathways underlying this cell-cycle effect. METHODS: Human umbilical venous endothelial cells were treated with Hcy, and/or LY294002, okadaic acid, peroxovanadate (PV), antisense Akt, phosphorylation of Akt and FKHRL1 proteins. p27(kip1) protein levels were measured with Western blotting, and Akt kinase activity and cell cycle were measured with immunoprecipitation and flow cytometry, respectively. RESULTS: We demonstrate that Hcy induces dephosphorylation of Akt and FKHRL1 and upregulates the cyclin-dependent kinase inhibitors p27(kip1) in a time- and dose-dependent manner. Phosphatidylinositol-3 kinase (PI3K) activator PV and phosphatase 2A inhibitor okadaic acid could reverse it, which suggests it was dependent on PI3K activity. Moreover, Hcy induces cell cycle G1 phase arrest prevented by pretreatment with PV and okadaic acid. Transfection with specific antisense oligonucleotides to Akt further proves the observations. CONCLUSIONS: The studies implied that a novel signaling pathway, PI3K/Akt/FOXO, might play an important role in mediating cell cycle G1 arrest in endothelial cells.     

The anti-proliferative inhibition of ellipticine in human breast mda-mb-231 cancer cells is through cell cycle arrest and apoptosis induction

Ellipticine, a cytotoxic plant alkaloid, is known to inhibit topoisomerase II. Here we report the mechanism of apoptosis induction and cell cycle arrest by ellipticine in human breast MDA-MB-231 cancer cells. Ellipticine treatment arrested MDA-MB-231 cells at the G2/M phase after 6 h of treatment. This effect was strongly associated with a concomitant decrease in the level of cyclin B1, Cdc25 and Cdc2, and increase in phospho-Cdc2 (Tyr15). In addition, ellipticine also induced apoptosis in MDA-MB-231 cells, as determined by using both DNA fragmentation and Annexin-V staining assay. Ellipticine increased the expression of Bax, but decreased the level of Bcl-2, Bcl-XL and X-linked inhibitor of apoptosis protein (XIAP), and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c, and activation of caspase-9 and -3). In addition, pre-treatment of cells with caspase-9 inhibitor inhibited ellipticine-induced cell proliferation and apoptosis, indicating that caspase-9 activation was involved in MDA-MB-231 cell apoptosis induced by ellipticine. Taken together, our study suggests that the inhibition of cell cycle progression signaling and initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of ellipticine in MDA-MB-231 cells.     

Euphorbia factor L2 induces apoptosis in A549 cells through the mitochondrial pathway

Euphorbia factor L2, a lathyrane diterpenoid isolated from caper euphorbia seed (the seeds of Euphorbia lathyris L.), has been traditionally applied to treat cancer. This article focuses on the cytotoxic activity of Euphorbia factor L2 against lung carcinoma A549 cells and the mechanism by which apoptosis is induced. We analyzed the cytotoxicity and related mechanism of Euphorbia factor L2 with an MTT assay, an annexin V-FITC/PI test, a colorimetric assay, and immunoblotting. Euphorbia factor L2 showed potent cytotoxicity to A549 cells. Euphorbia factor L2 led to an increase in reactive oxygen species (ROS) generation, a loss of mitochondrial electrochemical potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase, suggesting that Euphorbia factor L2 induced apoptosis through a mitochondrial pathway. The cytotoxic activity of Euphorbia factor L2 in A549 cells and the related mechanisms of apoptotic induction provide support for the further investigation of caper euphorbia seeds.     

Induction of cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells by casuarinin from the bark of Terminalia arjuna Linn

Casuarinin, a hydrolyzable tannin isolated from the bark of Terminalia arjuna Linn. (Combretaceae), inhibits human non-small cell lung cancer A549 cells by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest is due to p53-dependent induction of p21/WAF1. An enhancement in Fas/APO-1 and the two forms of Fas ligand (FasL), membrane-bound FasL and soluble FasL, might be responsible for the apoptotic effect induced by casuarinin. Our study reports here for the first time that the induction of p53 and the activity of the Fas/FasL apoptotic system may participate in the antiproliferative activity of casuarinin in A549 cells.     

Ethanol extract of Dunaliella salina induces cell cycle arrest and apoptosis in A549 human non-small cell lung cancer cells

The ethanol extract of Dunaliella salina (EDS) on proliferation and apoptosis in the A549 human lung cancer cell line and their associated protein expressions were investigated. After 24 and 48 h treatment, MTT assay showed that 25 microg/ml of EDS significantly reduced A549 cell proliferation by 25.2% (p<0.05) and 48.3% (p<0.01), respectively. To explore its molecular mechanisms in regulating cell proliferation, we first showed that EDS markedly reduced A549 proliferation via inhibition of BrdU incorporation at 25 microg/ml by 65.8% (p<0.001). By cytometric analysis, EDS was found to induce apoptosis and cell cycle arrest in the G0/G1 phase. In the DNA gel electrophoresis assay, EDS (25, 50 and 100 microg/ml) induced significant apoptosis at 48 h. Annexin V/Propodium iodide double staining demonstrated that administration of EDS (25 microg/ml) in 12, 24 and 48 h induces apoptosis of 27.7%, 30.7%, and 38.7%. Western blotting assay demonstrated that EDS significantly increased the expression of cyclin-dependent kinase (CDK) inhibitors p53 and p21 and death-receptor proteins Fas and FasL. Bax expression was also elevated by treatment with EDS. Our data suggested that EDS could influence the antiproliferative effects and induce cell cycle G0/G1 arrest and apoptosis of A549 lung cancer cells.     

Growth inhibitory effect of KYKZL-1 on Hep G2 cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G₂ growth. We found that KYKZL-1 inhibited the growth of Hep G₂ cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE₂ and LTB₄ production in Hep G₂ cells. Accordingly, exogenous addition of PGE₂ or LTB₄ reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S–G₂ checkpoint via the activation of p21^CIP1 protein and down-regulation of cyclin A expression.These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest.Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile.     

Dihydroxyacetone induces G2/M arrest and apoptotic cell death in A375P melanoma cells

The active ingredient in sunless tanning products (STPs) is a simple sugar, dihydroxyacetone (DHA). Several studies have demonstrated that DHA is absorbed within the viable layers of skin and not fully contained within the stratum corneum. Additionally, spray tanning and other aerosolized application methods have increased the risk of internal exposure through mucous membranes and inhalation. Beyond its presence in STPs, DHA also occurs as an endogenous by‐product of fructose metabolism, and an excess of DHA in cells can induce advanced glycation end (AGE) products and oxidative stress. Therefore, exogenous and endogenous exposures to DHA may be harmful to cells, and it has already been demonstrated that exogenous exposure to DHA is cytotoxic in immortalized keratinocytes. Still, little is known about the exogenous DHA exposure effects on other skin components. In this study, we explore the effects of exogenous DHA exposure in a human melanoma cell line, A375P. Melanoma cells were sensitive to DHA and displayed a transient burst of reactive oxygen species within an hour of exposure. Cell cycle arrest at G2/M was observed within 24 h of exposure, and apoptosis, monitored by the cleavage of PARP‐1 and Caspase‐3, was detected within 72 h of exposure to DHA. Together, these demonstrate that exogenous exposure to DHA has cytotoxic effects in our selected cell model and indicates the need to further investigate the exogenous exposure effects of DHA in other relevant exposure models.     

Quercetin-mediated cell cycle arrest and apoptosis involving activation of a caspase cascade through the mitochondrial pathway in human breast cancer MCF-7 cells

Dietary polyphenols have been correlated with a reduced risk of developing cancer. Quercetin (a natural polyphenolic compound) induced apoptosis in many human cancer cell lines, including breast cancer MCF-7 cells. However, the involvement of possible signaling pathways and the roles of quercetin in apoptosis are still undefined. The purpose of this study was to investigate the effects of quercetin on the induction of the apoptotic pathway in human breast cancer MCF-7 cells. When MCF-7 cells were treated with quercetin for 24 and 48 h and at various doses (10–175 μM), cell viability decreased significantly in time- and dose-dependent manners. Exposure of MCF-7 cells to 10–175 μM quercetin resulted in an approximate 90.25% decrease in viable cells. To explicate the mechanism underlying the antiproliferative effect of quercetin, cell cycle distribution and apoptosis in MCF-7 cells was investigated after exposure to 150 μM quercetin for 6–48 h. Quercetin caused a remarkable increase in the number of S phase (14.56% to 61.35%) and sub-G1 phase cells (0.1% to 8.32%) in a dose- and time-dependent manner. Quercetin caused S phase arrest by decreasing the protein expression of CDK2, cyclins A and B while increasing the p53 and p57 proteins. Following incubation with quercetin for 48 h, MCF-7 cells showed apoptotic cell death by the decreased levels of Bcl-2 protein and ΔΨ _m and increased activations of caspase-6, -8 and -9. Moreover, quercetin increased the AIF protein released from mitochondria to nuclei and the GADD153 protein translocation from endoplasmic reticulum to the nuclei. These data suggested that quercetin may induce apoptosis by direct activation of the caspase cascade through the mitochondrial pathway in MCF-7 cells.     

Artesunate enhances radiosensitivity of human non-small cell lung cancer A549 cells via increasing NO production to induce cell cycle arrest at G2/M phase

The resistance of non-small cell lung cancer (NSCLC) to radiation is the major reason for radiotherapy failure of this kind cancer. Currently, there is no effective radiosensitizer in clinical use. Artemisinin and its derivates enhance radiotherapeutic effect in some kinds of tumors; however, whether artemisinin and its derivates can enhance the radiosensitivity of NSCLC remains unknown. Therefore, in the present experiments, artemisinin and its derivatives were firstly screened for their radiosensitization on NSCLC A549 (A549) cells and then the possible mechanisms were investigated. Our results showed that artesunate enhance radiosensitivity of A549 cells in vitro among artemisinin and its derivatives, and artesunate combined with local radiotherapy retarded the tumor growth in nude tumor xenografts; the inhibition produced by 30 mg/kg of artesunate was 74.6%. The results on the possible mechanisms showed artesunate increased the NO level within irradiated A549 cells. Artesunate didn't induce apoptosis of irradiated cells but induced G₂/M arrest. The induced G₂/M arrest was related to down-regulated cyclin B1 mRNA expression. Taken together, artesunate exhibited potent radiosensitivity against human A549 cells in vitro and in vivo, probably via NO signal transduction pathway to induce cell cycle arrest at G₂/M phase. Therefore, artesunate should be further investigated as a radiosensitizer in clinical application.