Human Carcinoma Cell Line A549 Research Articles

Hydroxyl-substituted double Schiff-base condensed 4-piperidone/cyclohexanones as potential anticancer agents with biological evaluation

Novel hydroxyl-substituted double Schiff-base 4-piperidone/cyclohexanone derivatives, 3a–e, 4a–e, 5a–d, and 6a–c, were synthesized and fully characterized by 1H NMR, IR and elemental analysis. The cytotoxicity against human carcinoma cell lines A549, SGC7901, HePG2, HeLa, K562, THP-1 and non-malignant LO2 cell lines were evaluated. The results showed 4-piperidinone derivatives displayed better cytotoxicity than cyclohexanone derivatives, especially for 3,4,5-trihydroxyphenyl-substituted BAP 5c. The western blot and flow cytometry results proved 5c can effectively promote cell apoptosis through up-regulating Bax protein and down-regulating Bcl-2 protein expression. Molecular docking modes showed that 5c could reasonably bind to the active site of Bcl-2 protein through strong intermolecular hydrogen bonds and significant hydrophobic effect. In vivo, 5c can effectively suppress the growth of HepG2 xenografts without apparent body weight changes. This study indicates that 5c can be a potential anticancer agent for early treatment of liver cancers.

UV Light Stimulated Activation of Camkii in Human Epidermoid Carcinoma A431 Cells

Summary The Ca2+/calmodulin dependent kinase II (CaMKII) represents a family of serine/threonine kinases with diverse functions, implicated in various signalling pathways in the cell, especially in those controlling cellular proliferation and cellular death. The human epidermoid carcinoma cell line A431 is an established model for the study of the cell cycle and cancer-associated signalling, in which the role of CaMKII is not so well elucidated. Therefore, the present study aimed to investigate the role of CaMKII in the induction of apoptosis in the cell line A431 following ultraviolet (UV) irradiation and to determine the downstream events with a focus on the caspase cascade. For the latter purpose, we employed a method initially developed for the simultaneous determination of the activity of 10 different caspase enzymes. Following exposure to UV light, we observed activation of the activator caspase-8 followed by activation of the effector caspase-3, triggering apoptosis in the epidermoid carcinoma cell line A431. The results supported a pivotal role of caspases and demonstrated that a protein kinase cascade involving caspases-8 and -3 is the main pathway driven in UV-induced apoptosis in the human carcinoma cell line A431, and apoptotic stimuli triggering this pathway passed through activation of the kinase CaMKII.

Adamantane acid esters with alkoxyaryl alcohols: Synthesis, antiproliferative activity, and influence on microtubule network of tumor cells

Adamantaneacetic and adamantanecarboxylic acid esters containing 3-hydroxy-4-methoxybenzyl, 3,4,5-trimethoxybenzyl, or 5-(hydroxymethyl)-2-methoxyphenyl groups were synthesized as unusual analogs of natural antitumor and anti-tubulin agents combretastatin A-4 and 2-methoxyestradiol. The compounds were found to possess noticeable cytotoxicity to epithelial human carcinoma cell line A549 (EC50 = 4.3—81 μmol L–1). An ability to cause complete depolymerization of microtubule network of A549 cells was demonstrated for 5-(hydroxymethyl)-2-methoxyphenyl adamantan-1-ylacetate (6a) at a concentration of 100 μmol L–1. Ester 6a belongs to a new structural type, which is unusual for the ligands of the tubulin colchicine domain, and is an interesting lead compound for further structural optimization.

Single-Walled Carbon Nanotubes Downregulate Stress-Responsive Genes in Human Respiratory Tract Cells

Carbon nanotubes (CNTs) are currently key promising materials of nanotechnology. However, elucidation of the possible effects of CNTs on the respiratory tract is urgently needed. The present study aimed to clarify the effect of single-walled CNTs (SWCNTs) on the expression of stress-responsive genes, using primary cultured normal human bronchial epithelial (NHBE), diseased HBE (DHBE) cells, and the human carcinoma cell lines A549 and FaDu. Purified SWCNTs were applied at concentrations of 0.1 or 1.0 mg/mL for 6 h, and a polymerase chain reaction (PCR) array was conducted to examine 84 stress-responsive genes. NHBE cell exposure to SWCNTs resulted in global downregulation of genes involved in inflammation, oxidative stress, and apoptosis. Further analysis using DHBE cells and carcinoma cell lines indicated a similar trend, although differences in sensitivity were observed. Downregulation of stress-responsive genes may be involved in the mechanism by which stress response protects against lung injury.

ChemInform Abstract: Ferrocene—Indole Hybrids for Cancer and Malaria Therapy

AbstractReview: 58 refs.

Sodium Orthovanadate Affects Growth of Some Human Epithelial Cancer Cells (A549, HTB44, DU145)

Within the concentration range of 1-20 microM, orthovanadate (Na3VO4) demonstrated a time and dose-dependent inhibition of autocrine growth of the human carcinoma cell lines A549 (lung), HTB44 (kidney) and DU145 (prostate), as compared to appropriate controls (without Na3VO4). The investigation was conducted by two methods: staining with N-hexa-methylpararosaniline (crystal violet=CV) or bromide3-(4,5-dimethyltio-azo-2)-2,5-diphenyl-tetrazole (MTT). In 5, 10 and 20 microM of Na3VO4 in serum-free medium, the mean values of these two tests for A549 were approximately 40%, 45% or 65% as compared to the appropriate controls. HTB44 had the greatest opportunity (statistically insignificant) at lower vanadium concentrations (up to 10 microM), whereas at 20 microM growth inhibition of these cells was approximately 50% of the controls. DU145 showed approximately 33%, 65% and 98% growth inhibition for 5, 10 and 20 microM of Na3VO4, respectively Additionally, hypothetical curves obtained by a MANOVA test based on the CV results after 72 h incubation with Na3VO4 in serum-free medium, and an example of a time-dependent effect of Na3VO4 on A549 cells, were also presented. Sodium orthovanadate was also examined for its cytotoxic capabilities, especially its ability to induce tumor cell apoptosis; the results were compared with the effect of paclitaxel. The target cells were dyed by differential staining (HOECHST33258 and propidium iodide) after 3 h and 24 h (DU145) or 3 h and 72 h (A549) of incubation with the vanadium compound. Contrary to the two cancer cell lines (viable, apoptotic or necrotic in experimental conditions), the renal HTB44 cells were insensitive up to 15 microM Na3VO4 concentrations. After 3 h incubation with Na3VO4, both lung (A549) and prostate (DU145) cancer cells showed a slight but significant reduction in the percentage of viable cells, and an increased amount of apoptotic cells. In contrast to the lung cells, DU145 prostate cells after 24 h were more sensitive to paclitaxel than to sodium orthovanadate. In the case of lung cells, the time of incubation was prolonged (to 72 h) to allow for a study of the effect of orthovanadate in greater detail. After 72 h of incubation with Na3VO4 or paclitaxel, A549 showed a similar level of viable cells (25-32% of total cultured cells); however, the percentage of apoptotic cells was higher in the case of A549 cells--ca 36% for both drugs, but the concentration of Na3VO4 was significantly greater than paclitaxel levels.

Gene expression profiling of the human carcinoma cell line A-431 after 5-aminolevulinic acid-based photodynamic treatment

The photosensitizer protoporphyrin IX, endogenously accumulated from the precursor aminolevulinic acid (ALA) as well as other less photodynamically effective intermediates, is a successful agent in photodynamic therapy. Despite encouraging clinical results, the basic mechanisms leading to cell death are not fully understood. To elucidate these fundamentals, the alteration of the gene expression pattern in the squamous cell carcinoma cell line A-431 was studied at different time-points after photodynamic treatment with ALA by cDNA-array technique. Cells were incubated for 16 h with 100 microg/ml ALA and irradiated with a fluence of 3.5 J/cm(2) resulting in 50% survival until 8 h post treatment. RNA was isolated at 1.5, 3, 5 and 8 h post treatment and from 3 controls (untreated, light only and dark), radioactively labeled by reverse transcription with 33P-dCTP and hybridized onto macroarray PCR filters containing PCR products of 2135 genes, which were selected for relevance in carcinogenesis, stress response and signal transduction. Verification of observed expression changes was carried out by real-time RT-PCR. We found a strong induction of expression of the immediate early genes c-jun and c-fos as well as decreased expression of genes involved in proliferation such as myc, genes involved in apoptosis such as Fas associated via death domain (FADD) and the fibronectin gene for cell adhesion. An apoptosis induction rate of not more than 30% as proved by apoptosis detection assays and caused by PpIX localization in the membrane was reflected by the expression profile.

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells.

We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.

P27Kip1 overexpression causes apoptotic death of mammalian cells.

p27KiP1, a member of the Cip/Kip family of cyclin-dependent kinase (cdk) inhibitors, has been implicated in mediating G1 arrest in response to a variety of growth inhibitory signals. Its importance in regulating cell growth is emphasized by the fact that mice lacking p27Kip1 are abnormally large and display hyperplasia of multiple tissues. However, these mice retain the ability to undergo G1 arrest in response to growth inhibitory signals, suggesting that p27KiP1 may serve other functions important for controlling tissue growth. In the present study, we utilized an adenoviral vector-based expression system to examine the consequences of p27Kip1 overexpression in the human carcinoma cell lines A549, HeLa and RKO, in human melanoma SK-MEL-110 cells, in human lung fibroblasts IMR90 and in the rat fibroblast line Rat1. We demonstrate that overexpression of p27Kip1 leads to apoptotic cell death in all cell types, and further show that ectopic expression of Bcl-2 can protect HeLa cells from apoptosis mediated by p27Kip1 overexpression. To our knowledge, this is the first study demonstrating that p27Kip1 can induce apoptosis. Our findings provide new insight into the possible functions of this growth regulatory protein, and support the potential utility of gene therapeutic approaches aimed at elevating p27Kip1 expression for treatment of human cancers.

Antiproliferative Activity of 3-Aminobenzamide in A431 Carcinoma Cells Is Associated with a Target Effect on Cytoskeleton

3-Aminobenzamide (3-ABA) is an inhibitor of poly(ADP-ribose)polymerase (PARP), an enzyme involved in several cellular processes, and exerts its effects by acting at the cytoskeleton level. Here we show that 3-ABA has an antiproliferative effect on the human carcinoma cell line A431, as measured by different assays. 3-ABA was capable of inhibiting cell growth as well as colony formation, this inhibitory effect is reversible. Morphological analyses showed a series of cellular alterations, such as a remarkable increase of dendritic-like protrusions, quite unusual in epithelial cells, and suggestive of a differentiative triggering. Immunocytochemical studies suggested that a major target of 3-ABA was indeed the cytoskeleton. These data, together with those of the literature, indicate that 3-ABA, depending on cell histotype and drug concentration, is a versatile drug capable of exerting antiproliferative and cytostatic effects as well as cytotoxic and antiapoptotic effects, processes sharing an important involvement of cytoskeleton. These unique characteristics of 3-ABA may be of interest for cancer research.

Immunohistochemical localization of cell surface receptors using a novel method permitting simple, rapid and reliable LM/EM correlation.

A method is presented which allows correlative serial section analysis by light and electron microscopy of cell surface antigens in monolayer cultures. Sites of antigenicity are shown by deposition of diaminobenzidine after pre-embedding, immunoperoxidase immunocytochemistry. Osmication is replaced by the use of gold chloride which specifically enhances the electron density of diaminobenzidine. In addition gold chloride bound to diaminobenzidine survives embedding and provides the basis for a post-embedding photochemical amplification method. Immunostained cells are embedded in LR White by a rapid technique which preserves their structure and leaves them available for subsequent post-embedding immunocytochemistry. The method is illustrated by the demonstration of epidermal growth factor (EGF) receptors on the EGF receptor-rich human carcinoma cell line A431 using a well characterized monoclonal antibody raised against EGF receptor.

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells.

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.

The human carcinoma cell line A431 possesses large numbers of functional β-adrenergic receptors

The existence of β-adrenergic receptors was demonstrated on whole A431 cells as well as A431 membrane preparations by means of binding assays using the hydrophobic 1-[ 3H]dihydroalprenolol and the hydrophilic antagonist [ 3H]CGP-12,177 as β-adrenergic ligands. Binding was stereospecific. The receptors, as shown by competition studies, proved to be of the β 2-subtype and appeared functional in the stimulation of adenylate cyclase. The number of receptors per cell and the yield of receptor sites/mg membrane protein render the A431 cell a useful tool for the study of human β-adrenergic receptors.