Vanadate-activated calcium influx in A431 cells is dependent on the plasma membrane potential

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two- to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down-regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium-independent and is not activated by depolarization in high KCl. On the contrary, vanadate-stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about -65 to -30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.     

Growth control of A431 cells in protein-free medium: Secretory products do not affect cell growth

Summary A431 cells grew at similar rates in protein-free Coon's modified Ham's F12 medium (PF-C-F12) with and without added bovine calf serum. The cells secreted a heparin-binding growth factor and a type-β transforming growth factor, but their growth in PF-C-F12 was not affected by these factors, or by DNA synthesis factor from Rhodamine fibrosarcoma, basic fibroblast growth factor, insulin, human transferrin, bovine serum albumin, and their combinations. Growth of A431 cells in PF-C-F12 was not density dependent and was not affected by either addition of conditioned medium or replacement of conditioned medium by fresh medium. These results indicate that A431 cells have an intracellular mechanism for autonomous growth, and that their growth is not affected by factors that they secrete or by exogenous growth factors. This work was supported by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture of Japan, and a grant from Hokkoku Cancer Research Foundation.     

Swertia chirayita suppresses the growth of non-small cell lung cancer A549 cells and concomitantly induces apoptosis via downregulation of JAK1/STAT3 pathway

Lung carcinoma is the leading cause of cancer-related mortalities worldwide, and present therapeutical interventions are not successful enough to treat this disease in many cases. Recent years have witnessed a surge in exploring natural compounds for their antiproliferative efficacy to expedite the characterization of novel anticancer chemotherapeutics. Swertia chirayita is a valued medicinal herb and possess intrinsic pharmaceutical potential. However, elucidation of its anticancer effects at molecular levels remains unclear and needs to be investigated. We assessed the anticancer and apoptotic efficacy of S. chirayita ethanolic extract (Sw-EtOH) on non-small cell lung cancer (NSCLC) A549 cells during this exploratory study. The results elucidated that S. chirayita extract induced toxic effects within lung cancer cells by ~1 fold during cytotoxicity and LDH release assay at a 400 μg/ml concentration. Sw-EtOH extract elevates the level of ROS, resulting in the disruption of Δψm and release of cytosolic cytochrome c by 3.15 fold. Activation of caspases-3, -8 & -9 also escalated by ~1 fold, which further catalyze the augmentation of PARP cleavage (~3 folds), resulting in a four-fold increase in Sw-EtOH induced apoptosis. The gene expression analysis further demonstrated that Sw-EtOH extracts inhibited JAK1/STAT3 signaling pathway by down-regulating the levels of JAK1 and STAT3 to nearly half a fold. Treatment of Sw-EtOH modulates the expression level of various STAT3 associated proteins, including Bcl-XL, Bcl-2, Mcl-1, Bax, p53, Fas, Fas-L, cyclinD1, c-myc, IL-6, p21 and p27 in NSCLC cells. Thus, our study provided a strong impetus that Sw-EtOH holds the translational potential of being further evaluated as efficient cancer therapeutics and a preventive agent for the management of NSCLC.     

芳姜黄酮对人皮肤鳞状细胞癌A431细胞迁移、侵袭及凋亡的影响和机制研究

为研究芳姜黄酮(Ar-Turmerone)对人鳞状细胞癌A431细胞增殖、迁移、侵袭和凋亡的影响及机制。实验采用CCK-8法检测抑制率,吉姆萨染色观察细胞形态,划痕实验和Transwell小室实验研究细胞迁移和侵袭能力的变化,流式细胞仪检测细胞凋亡率。此外,通过实时荧光定量聚合酶链反应(Real-time PCR)与蛋白质印迹法(western blot)法检测mRNA和蛋白表达。siRNA阻断Notch1,Hes1和PTEN,检测相应的下游mRNA和蛋白的表达变化,流式细胞仪检测细胞凋亡率。结果发现,芳姜黄酮可以抑制A431细胞增殖,使细胞形态发生改变,抑制细胞体外迁移和侵袭能力,促进细胞凋亡。经过芳姜黄酮处理后,Notch1,Hes1,PTEN的mRNA和蛋白表达升高。沉默Notch1,Hes1 mRNA和蛋白表达低于单纯给药组,而沉默Hes1,PTEN mRNA和蛋白表达也低于单纯给药组;沉默PTEN后,与单纯给药组相比,细胞死亡率降低。总之,芳姜黄酮可以抑制人鳞状细胞癌A431细胞的增殖并促进其凋亡,且具有抑制体外迁移和侵袭的作用,其促进细胞凋亡的机制是通过Notch1/Hes1/PTEN途径实现的。     

Tyrosin dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3α by genistein in A431 cells

Modulation of protein Kinase _F/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F _A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could become tyrosine-dephosphorylated and inactivated down to less than 30% of control values in concentration dependent manner by 50-400 μM genistein( a Specific tyrosine kinase inhibitor), as demonstrated by metobolic ³²p-labeling of the cells followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and byimmunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Taken together, the results provide evidence that Kinase F_A/GSK-3α may exist in a highly tyrosine-phosphorylated/activated state in resting cells which can by tyrosine-dephosphorylated and nactivated by extracellular stimulus and that tyrosine kinase(s) and /or tyrosine phosphatase(s) may play a role in the modulation of kinse F_A/GSK-3α activity in cells.     

Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21^Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process. J. Cell. Biochem. 70:442-454, 1998. © 1998 Wiley-Liss, Inc.     

Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylatioin/ inactivation of protein kinase FA/GSK-3α in A431 cells

The signal transducrion mechanism of protein kinase F_A/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F_A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated to ～60% of the control level when cells were acutely treated with 1 μM tumor phorbol ester (TPA) at 37^oC for 30 min, as demonstrated by metabolic ³²P-labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 μM TPA at 37°C for 24 h and processed under identical condetions, kinase F_A/GSK-3α was found to be rephosphorylated on tyrosine residue and reactivated to ～130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase F_A/GSK-3α can be modulated in a reversible manner by short-term and long-term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up-regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down-regulation of PKC, the results further suggest that the TPA-mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.     

Temperature-dependent lateral and transverse distribution of the epidermal growth factor receptor in A431 plasma membranes

Summary To elucidate further the structure and molecular dynamics of the epidermal growth factor receptor, temperature-dependent aggregation and extracellular protrusion of the epidermal growth factor receptor in isolated plasma membranes from A431 cells were examined by fluorescence energy-transfer techniques. Epidermal growth factor was labeled at the amino terminus with either fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. A radionuclide receptor displacement assay demonstrated the bioactivity of these derivatives. Aggregation of the epidermal growth factor receptor was measured by determining the increase in fluorescence energy transfer between receptorbound fluorescein and tetramethylrhodamine-labeled epidermal growth factor. Energy transfer between receptor-bound fluorescent derivatives was reversibly greater at 37 than 4°C, indicating temperature-dependent aggregation of the receptor. The extracellular protrusion of the epidermal growth factor receptor was calculated from the magnitude of energy transfer between receptorbound fluorescein labeled epidermal growth factor and 5-(N-dodecanoylamino)-eosin partitioned into the lipid membrane at 4 and 37°C. No significant change in the distance of closest approach between the N-terminus of epidermal growth factor and the plasma membrane was observed at 4°C (69±2 Å) and 37°C (67±2 Å). Thus, the extracellular protrusion of the occupied epidermal growth factor receptor did not change detectably upon receptor aggregation.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

Tilianin inhibits the human ovarian cancer (PA-1) cell proliferation via blocking cell cycle,inducing apoptosis and inhibiting JAK2/STAT3 signaling pathway

Ovarian cancer is one of the deadliest gynecologic malignancies and is the seventh leading cause of mortalities and morbidities globally. Although there are various therapeutic strategies, a major challenge for scientific community is to come up with effective strategy to treat ovarian cancer. Tilianin, a polyphenol flavonoid is well known for its extensive biological actions like cardioprotective, neuroprotective, anti-oxidant, anti-inflammatory, anti-diabetic and anti-tumor properties. The current study is designed to investigate the anti-cancer action of Tilianin in ovarian cancer (PA-1) cells. The findings of this study revealed that Tilianin treatment results in significant and concentration dependent decrease in cell viability. The growth inhibiting action of Tilianin is associated with apoptosis which was confirmed by DAPI and AO/EtBr staining. The Tilianin-triggered apoptosis in PA-1 cells was correlated with elevated generation of ROS, loss of mitochondrial membrane potential, alterations in pro-apoptotic (upregulated mRNA expression of Bax) and anti-apoptotic (downregulated mRNA expression of Bcl2) factors and activation of caspase-8, −9 and −3. Cell cycle analysis revealed that Tilianin treatment prevented G1/S transition through reduced mRNA expression of cyclin D1. Additionally, the findings of this study also showed Tilianin inhibited JAK2/STAT3 signaling (downregulated expression of pJAK2, JAK2, pSTAT3, and STAT3) with no change in mRNA expression level of ERK indicating its non-involvement in the apoptotic and/or growth inhibition of ovarian cancer cells. In conclusion, the findings of this exploration provided clear evidence of anti-cancer effects of Tilianin in PA-1 cells through its anti-proliferative action, ability to induce apoptosis both through extrinsic and intrinsic pathways, cell cycle (G1/S) arrest and JAK2/STAT3 signaling inhibition.     

Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.     

The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade

Despite the involvement of the elongation factors eEF1A (eEF1A1 and eEF1A2) in the development of different cancers no information is available on their possible contribution to the biology of hepatocellular carcinoma (HCC). We investigated the expression of both forms of eEF1A in HepG2 and JHH6 cell lines considered to be a good in vitro model of HCC at different stage of differentiation. Our data indicate that the mRNA amount of eEF1A1 is increased in both cell lines as compared to normal liver tissue, but eEF1A2 mRNA level is markedly increased only in JHH6. Moreover, the less differentiated cell line JHH6 displays higher EEF1A1 and EEF1A2 mRNAs levels and an higher nuclear-enriched/cytoplasm ratio of EEF1A protein compared to the better differentiated HepG2 cell line. Over-expression depends only partially on gene amplification. The more abundant mRNA levels and the higher nuclear-enriched/cytoplasm ratio of eEF1A in JHH6 neither correlate with apoptosis resistance nor with proliferation rate in sub-confluent cells. However, in confluent cells, a clear tendency to maintain JHH6 into the cell cycle was observed. In conclusion, we document the increased mRNA levels of EEF1A genes in HCC cell lines compared to normal liver. Additionally, we show the increased nuclear-enriched/cytoplasmic protein ratio of eEF1A and the marked raise of the expression of both eEF1A forms in JHH6 compared to HepG2, suggesting the possibility that eEF1A forms might become a relevant markers related to HCC tumor phenotype.