Epidermoid Carcinoma Cell Line A431 Research Articles

ΔM4: Membrane-Active Peptide with Antitumoral Potential against Human Skin Cancer Cells.

Peptides have become attractive potential agents due to their affinity to cancer cells. In this work, the biological activity of the peptide ΔM4 against melanoma cancer cell line A375, epidermoid carcinoma cell line A431, and non-tumoral HaCaT cells was evaluated. The cytotoxic MTT assay demonstrates that ΔM4 show five times more activity against cancer than non-cancer cells. The potential membrane effect of ΔM4 was evaluated through lactate dehydrogenase release and Sytox uptake experiments. The results show a higher membrane activity of ΔM4 against A431 in comparison with the A375 cell line at a level of 12.5 µM. The Sytox experiments show that ΔM4 has a direct effect on the permeability of cancer cells in comparison with control cells. Infrared spectroscopy was used to study the affinity of the peptide to membranes resembling the composition of tumoral and non-tumoral cells. The results show that ΔM4 induces a fluidization effect on the tumoral lipid system over 5% molar concentration. Finally, to determine the appearance of phosphatidylserine on the surface of the cell, flow cytometry analyses were performed employing an annexin V-PE conjugate. The results suggest that 12.5 µM of ΔM4 induces phosphatidylserine translocation in A375 and A431 cancer cells. The findings of this study support the potential of ΔM4 as a selective agent for targeting cancer cells. Its mechanism of action demonstrated selectivity, membrane-disrupting effects, and induction of phosphatidylserine translocation.

Dimeric Benzodiazepines as Peptide Mimetics to Overcome p53-Dependent Drug Resistance of Tumors.

Benzodiazepines that consist of one α- and one β-amino acid residues linked together in a seven-membered heterocyclic ring could be treated as small, rigid, cyclic dipeptides capable of exhibiting a wide range of biological activities. During our research on novel analogues of anthramycin, a tricyclic antibiotic benzodiazepine, we developed the synthesis of two benzodiazepine dimers, obtained through the cyclization of appropriate linear tripeptides. The synthesized compounds were tested on a panel of seven cancer and normal cell lines. The developed molecules exhibited promising cytotoxic activity against the lung cancer cell lines A549 and NCI-H1299 and the epidermoid carcinoma cell line A-431. Moreover, they showed significant selectivity compared to the reference cell lines (BJ-human normal skin fibroblasts and MRC-5-human normal lung cell line). When tested on two isogenic cell lines, HCT116 and HCT116p53-/- (colon cancer), contrary to cisplatin being used as a positive control, the obtained compounds showed a cytotoxic effect independent of the p53 protein status. For the above reasons, the obtained compounds can be considered a new group of promising anticancer agents, useful in the fight against p53-dependent drug resistance in cancers. They can also be treated as convenient, leading structures suitable for further optimization and searching for more active and selective molecules.

Chemical Characteristics of Ethanol and Water Extracts of Black Alder (Alnus glutinosa L.) Acorns and Their Antibacterial, Anti-Fungal and Antitumor Properties.

The aim of this study was to identify polyphenolic compounds contained in ethanol and water extracts of black alder (Alnus glutinosa L.) acorns and evaluate their anti-cancer and antimicrobial effects. The significant anti-cancer potential on the human skin epidermoid carcinoma cell line A431 and the human epithelial cell line A549 derived from lung carcinoma tissue was observed. Aqueous and ethanolic extracts of alder acorns inhibited the growth of mainly Gram-positive microorganisms (Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans) and yeast-like fungi (Candida albicans, Candida glabrata), as well as Gram-negative (Escherichia coli, Citrobacter freundii, Proteus mirabilis, Pseudomonas aeruginosa) strains. The identification of polyphenols was carried out using an ACQUITY UPLC-PDA-MS system. The extracts were composed of 29 compounds belonging to phenolic acids, flavonols, ellagitannins and ellagic acid derivatives. Ellagitannins were identified as the predominant phenolics in ethanol and aqueous extract (2171.90 and 1593.13 mg/100 g DM, respectively) The results may explain the use of A. glutinosa extracts in folk medicine.

Wet chemical development of CuO/GO nanocomposites: its augmented antimicrobial, antioxidant, and anticancerous activity

This study employed a bottom-up technique to synthesize copper oxide (CuO) nanoparticles over hydrophilic graphene oxide (GO) nanosheets. The CuO/GO nanocomposite has been prepared using two selected precursors of copper nitrate and citric acid with an intermittent mixing of GO solutions. The synthesized Nanocomposites were characterized using different biophysical techniques like FT-IR, NMR, FE-SEM, and HR-TEM analyses. FT-IR analyses confirm the nanocomposites’ successful formation, which is evident from the functional groups of C=C, C-O, and Cu-C stretching vibrations. Morphological analyses reveal the depositions of CuO nanoparticles over the planar rough GO sheets, which has been elucidated from the FE-SEM and HR-TEM analyses supported by respective EDAX analyses. The antimicrobial activities have been evident from the surface roughness and damages seen from the FE-SEM analyses. The CuO/GO sheets were tested against Gram-positive (e.g., Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa). It is evident that the intrinsic antibacterial activity of CuO/GO sheets, when combined in equal proportions, elicited a robust antibacterial activity when tested over Gram –ve representative bacteria Escherichia coli. The antioxidant behaviour of synthesized CuO/GO nanocomposite was evaluated by scavenging the free radicals of DPPH and ABTS. Moreover, the cytotoxic activity was also studied against epidermoid carcinoma cell line A-431. A brief mathematical formulation has been proposed in this study to uncover the possibilities of using the nanocomposites as potential drug candidates in theranostic applications in disease treatment and diagnosis. This study would help uncover the electronic properties that play in the nano-scaled system at the material-bio interface, which would aid in designing a sensitive nano-electromechanical device bearing both the therapeutic and diagnostic attributes heralding a new horizon in the health care systems.

Costunolide, a Sesquiterpene Lactone, Suppresses Skin Cancer via Induction of Apoptosis and Blockage of Cell Proliferation.

Costunolide is a naturally occurring sesquiterpene lactone that demonstrates various therapeutic actions such as anti-oxidative, anti-inflammatory, and anti-cancer properties. Costunolide has recently emerged as a potential anti-cancer agent in various types of cancer, including colon, lung, and breast cancer. However, its mode of action in skin cancer remains unclear. To determine the anti-cancer potential of costunolide in skin cancer, human epidermoid carcinoma cell line A431 was treated with costunolide. A lactate dehydrogenase assay showed that costunolide diminished the viability of A431 cells. Apoptotic cells were detected by annexin V/propidium iodide double staining and Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay assay, and costunolide induced cell apoptosis via activation of caspase-3 as well as induction of poly-ADP ribose polymerase cleavage in A431 cells. In addition, costunolide elevated the level of the pro-apoptotic protein Bax while lowering the levels of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. To address the inhibitory effect of costunolide on cell proliferation and survival, various signaling pathways, including mitogen-activated protein kinases, signal transducer and activator of transcription 3 (STAT3), nuclear factor κB (NF-κB), and Akt, were investigated. Costunolide activated the p38 and c-Jun N-terminal kinase pathways while suppressing the extracellular signal-regulated kinase (ERK), STAT3, NF-κB, and Akt pathways in A431 cells. Consequently, it was inferred that costunolide suppresses cell proliferation and survival via these signaling pathways. Taken together, our data clearly indicated that costunolide exerts anti-cancer activity in A431 cells by suppressing cell growth via inhibition of proliferation and promotion of apoptosis. Therefore, it may be employed as a potentially tumor-specific candidate in skin cancer treatment.

Low-frequency mechanical vibration induces apoptosis of A431 epidermoid carcinoma cells.

Cancer research is increasingly focused on discovering strategies to induce cancer cell apoptosis without affecting surrounding normal cells. One potential biocompatible method is mechanical vibration, which has been developed as part of the emerging field of mechanomedicine. Previous studies of mechanical vibration have employed high‐frequency vibration, which damages healthy cells. In this study, we examined the effects of brief (1 h) low‐frequency (20 Hz) mechanical vibration on glucose consumption and survival (apoptosis, necrosis, HMGB1 release) of the human epidermoid carcinoma cell line A431. We found that apoptosis, but not necrosis, was significantly increased at 48 h after mechanical vibration compared with cells maintained in static culture. In keeping with this, extracellular release of HMGB1, a necrosis marker, was lower in cultures of A431 cells subjected to mechanical vibration compared with control cells. Glucose consumption was increased in the first 24 h after mechanical vibration but returned to control levels before the onset of apoptosis. Although the precise intracellular mechanisms by which low‐frequency mechanical vibration triggers apoptosis of A431 cells is unknown, these results suggest a possible role for metabolic pathways. Mechanical vibration may thus represent a novel application of mechanomedicine to cancer therapy.

Способ получения экстрактов с противоопухолевой активностью из Daedaleopsis confragosa К-1326

A method for extraction and fractionation of proteins and polysaccharides from the biomass of the basidial fungus Daedaleopsis confragosa K-1326, having antitumor activity, shown on the epidermoid carcinoma cell line A431 and the cells of melanoma В16, has been developed. This method allows for reproducible production of series of preparations and can be used to obtain preparations from other basidiomycetes. The results obtained in this work show the promising prospects for the development and use of drugs from basidiomycetes for treatment of cancer. Basidiomycetes, Daedaleopsis confragosa, antitumor activity, fractionation, extraction The work was performed under the State Assignment «Development of preventive and therapeutic drugs based on basidiomycetes for the prevention and treatment of influenza with pandemic potential».

MAPK/ERK regulation of P53 in human epidermoid carcinoma cell line A431

Objective: To observe the impact of activation and inhibition of mitogen activated protein kinases (MAPK)/extracellular signalregulated protein kinase (ERK) signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma (SCC). cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC. Methods: Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-, medium- and highconcentration of inhibitors (PD98059 + DMSO), MAPK/ERK activation groups with low-, medium- and high-concentration of stimuli (IGF + PBS) and blank control group (DMSO). The cell proliferation in vitro was detected by MTT assay, with the cell apoptosis detected by flow cytometry (FCM) and the protein expression of P-ERK and P53 detected by western blot in each group. Results: The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration - effect and time - effect relationship ( p < .05); and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration - effect and time - effect relationship ( p < .05). The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059, with a clear concentration - effect relationship ( p < .05); while the apoptosis rate was decreased significantly after A431 cells were treated with IGF, also with a concentration - effect relationship ( p < .05). The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059. With the concentration of PD98059 going up, the decrease in P-ERK and the increase in P53 were more significant ( p < .05); while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF. With the concentration of IGF going up, the increase in P-ERK and the decrease in P53 were more significant ( p < .05). According to Pearson correlation analysis, the expression of P53 was negatively correlated to that of P-ERK ( p < .05). Conclusions: After MAPK/ERK signaling pathway was activated by IGF in A431 cells, the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced. However, after the inhibition of MAPK/ERK signaling pathway, the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.

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.

PO-231 mTOR activity differences and related metabolic activity in human breast cancer cell lines

IntroductionV-ATPase is a family of proton pumps that balances the proton between intracellular and extracellular compartments. High metabolism could be found in cancer cells resulting in overproduction of protons. Thus, highly activated V-ATPase is necessary to balance cytosolic pH in cancer cells to protect cell death from acidosis. As V-ATPase is important for cancer cell survivor, invasion, migration, and metastasis in cancer cells, therefore the development of anti-cancer therapeutics selectively target V-ATPases function in cancer cells is necessary.Material and methodsTo investigate the effect of C. cochinchinense extract on V-ATPase inhibition, the cancer cells were treated with C. cochinchinense for 24 hour then were stained with acridine orange for 30 min compared with a positive control (bafilomycin A1). Then, the effect of V-ATPase inhibition on apoptosis cell death induction was conducted by staining with Hoechst 33 342 to determine nuclear morphological changes.Results and discussionsThe results showed that C. cochinchinense extract inhibited V-ATPase activity in malignant melanoma cell line A375, triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell line HCT116 and epidermoid carcinoma cell line A431 compared with bafilomycin A (as a V-ATPase inhibitor). In addition, C. cochinchinense extract showed apoptosis induction by inducing apoptotic bodies in C. cochinchinense extract treated cells.ConclusionOur results indicated that C. cochinchinense extract is an inhibitor of V-ATPase triggering apoptosis induction in various cancer cell lines including A431, A375, and MDA-MB-231 cells. Thus C. cochinchinense extract may be further developed as an anti-cancer drug possessing V-ATPase inhibition. However, the underlying mechanisms of V-ATPase inhibitor and apoptosis induction in cancer cells should be further studied.

PO-230 Effect of C. cochinchinense extract on V-ATPase inhibition triggering apoptosis in human cancer cell lines

Introduction V-ATPase is a family of proton pumps that balances the proton between intracellular and extracellular compartments. High metabolism could be found in cancer cells resulting in overproduction of protons. Thus, highly activated V-ATPase is necessary to balance cytosolic pH in cancer cells to protect cell death from acidosis. As V-ATPase is important for cancer cell survivor, invasion, migration, and metastasis in cancer cells, therefore the development of anti-cancer therapeutics selectively target V-ATPases function in cancer cells is necessary. Material and methods To investigate the effect of C. cochinchinense extract on V-ATPase inhibition, the cancer cells were treated with C. cochinchinense for 24 hour then were stained with acridine orange for 30 min compared with a positive control (bafilomycin A1). Then, the effect of V-ATPase inhibition on apoptosis cell death induction was conducted by staining with Hoechst 33 342 to determine nuclear morphological changes. Results and discussions The results showed that C. cochinchinense extract inhibited V-ATPase activity in malignant melanoma cell line A375, triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell line HCT116 and epidermoid carcinoma cell line A431 compared with bafilomycin A (as a V-ATPase inhibitor). In addition, C. cochinchinense extract showed apoptosis induction by inducing apoptotic bodies in C. cochinchinense extract treated cells. Conclusion Our results indicated that C. cochinchinense extract is an inhibitor of V-ATPase triggering apoptosis induction in various cancer cell lines including A431, A375, and MDA-MB-231 cells. Thus C. cochinchinense extract may be further developed as an anti-cancer drug possessing V-ATPase inhibition. However, the underlying mechanisms of V-ATPase inhibitor and apoptosis induction in cancer cells should be further studied.

11C]Erlotinib PET cannot detect acquired erlotinib resistance in NSCLC tumor xenografts in mice

Introduction[11C]Erlotinib PET has shown promise to distinguish non-small cell lung cancer (NSCLC) tumors harboring the activating epidermal growth factor receptor (EGFR) mutation delE746-A750 from tumors with wild-type EGFR. To assess the suitability of [11C]erlotinib PET to detect the emergence of acquired erlotinib resistance in initially erlotinib-responsive tumors, we performed in vitro binding and PET experiments in mice bearing tumor xenografts using a range of different cancer cells, which were erlotinib-sensitive or exhibited clinically relevant resistance mechanisms to erlotinib. MethodsThe following cell lines were used for in vitro binding and PET experiments: the epidermoid carcinoma cell line A-431 (erlotinib-sensitive, wild-type EGFR) and the three NSCLC cell lines HCC827 (erlotinib-sensitive, delE746-A750), HCC827EPR (erlotinib-resistant, delE746-A750 and T790M) and HCC827ERLO (erlotinib-resistant, delE746-A750 and MET amplification). BALB/c nude mice with subcutaneous tumor xenografts underwent two consecutive [11C]erlotinib PET scans, a baseline scan and a second scan in which unlabeled erlotinib (10mg/kg) was co-injected. Logan graphical analysis was used to estimate total distribution volume (VT) of [11C]erlotinib in tumors. ResultsIn vitro experiments revealed significantly higher uptake of [11C]erlotinib (5.2-fold) in the three NSCLC cell lines as compared to A-431 cells. In all four cell lines co-incubation with unlabeled erlotinib (1μM) led to significant reductions in [11C]erlotinib uptake (−19% to −66%). In both PET scans and for all four studied cell lines there were no significant differences in tumoral [11C]erlotinib VT values. For all three NSCLC cell lines, but not for the A-431 cell line, tumoral VT was significantly reduced following co-injection of unlabeled erlotinib (−20% to −35%). ConclusionsWe found no significant differences in the in vitro and in vivo binding of [11C]erlotinib between erlotinib-sensitive and erlotinib-resistant NSCLC cells. Our findings suggest that [11C]erlotinib PET will not be suitable to distinguish erlotinib-sensitive NSCLC tumors from tumors with acquired resistance to erlotinib.

PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a positron-emitting radionuclide 89Zr. The 89Zr-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160±60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, 89Zr-DFO-ZEGFR:2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6±0.5% ID/g and tumour-to-blood ratio of 3.7±0.6 at 24 h after injection. 89Zr-DFO-ZEGFR:2377 provides higher tumour-to-organ ratios than anti-EGFR antibody 89Zr-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using 89Zr-DFO-ZEGFR:2377 at both 3 and 24 h after injection. In conclusion, 89Zr-DFO-ZEGFR:2377 is a potential probe for PET imaging of EGFR-expression in vivo.

Folate-Conjugated pH-Responsive Nanocarrier Designed for Active Tumor Targeting and Controlled Release of Gemcitabine.

The prime end of this study was to design a novel pH-sensitive as well as a PEGylated dendritic nanocarrier for both controllable and traceable gemcitabine delivery to cancerous cells. To accomplish this goal, we took advantage of a hybrid of nanoparticles including: mesoporous silica, graphene oxide and magnetite. The nanocarrier was prepared in a multi-step synthesis route. First, magnetite mesoporous silica was deposited on the graphene oxide matrix. Then, polyamidoamine dendrimers (up to generation 1.5) with pentaethylene hexamine end groups were grafted on the surface of the nanoparticles. In order to enhance the biostability, and as the next step, the nanocarrier was modified by polyethylene glycol. Finally, these particles were functionalized by folic acid as tumor targeting agents. According to the dynamic light scattering results, the hydrodynamic diameter of magnetic mesoporous silica graphene oxide hybrid nanoparticle was 152 ± 3nm, while for the supramolecular hybrid nanoparticles it was about 324 ± 12nm. Attained through the adsorption branch, the average pore diameter of these nanoparticles was 7.6nm. Zeta potential test indicated -27.1mV value for hybrid nanoparticles and +7.35mV for supramolecular hybrid nanoparticles. Besides, cytotoxicity assay showed enhanced cytotoxicity of epidermoid carcinoma cell line A431 in the presence of folate conjugated carriers. The maximum release occurred at the pH5.5, because the dendritic structure was in the open state rather than compact state. The enhanced cytotoxicity of the epidermoid carcinoma cell line A431 in the presence of folate conjugated carriers, confirmed the improved cancerous cells uptake. Also, the positive surface potential would be a good property for the biological applications because the inherent negative-charged surface of cell membranes facilitates the uptake of positive particles by electrostatic interactions.

Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma.

Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431

Hypoxia is a critical microenvironmental factor that drives cancer progression through angiogenesis and metastasis. Glycoproteins, especially those on the plasma membrane, orchestrate this process; however, questions remain regarding hypoxia-perturbed protein glycosylation in cancer cells. We focused on the effects of hypoxia on the integrin family of glycoproteins, which are central to the cellular processes of attachment and migration and have been linked with cancer in humans. We employed electrostatic repulsion hydrophilic interaction chromatography coupled with iTRAQ labeling and LC-MS/MS to identify and quantify glycoproteins expressed in A431. The results revealed that independent of the protein-level change, N-glycosylation modifications of integrin α 3 (ITGA3) were inhibited by hypoxia, unlike in other integrin subunits. A combination of Western blot, flow cytometry, and cell staining assays showed that hypoxia-induced alterations to the glycosylation of ITGA3 prevented its efficient translocation to the plasma membrane. Mutagenesis studies demonstrated that simultaneous mutation of glycosites 6 and 7 of ITGA3 prevented its accumulation at the K562 cell surface, which blocked integrin α 3 and β 1 heterodimer formation and thus abolished ITGA3's interaction with extracellular ligands. By generating A431 cells stably expressing ITGA3 mutated at glycosites 6 and 7, we showed that lower levels of ITGA3 on the cell surface, as induced by hypoxia, conferred an increased invasive ability to cancer cells in vitro under hypoxic conditions. Taken together, these results revealed that ITGA3 translocation to the plasma membrane suppressed by hypoxia through inhibition of glycosylation facilitated cell invasion in A431.

Isolation, cytotoxicity evaluation and HPLC-quantification of the chemical constituents from Prangos pabularia.

Phytochemical analysis of the dichloromethane:methanol (1∶1) extract of root parts of Prangos pabularia led to the isolation of twelve cytotoxic constituents, viz., 6-hydroxycoumarin (1), 7-hydroxycoumarin (2), heraclenol-glycoside (3), xanthotoxol (4), heraclenol (5), oxypeucedanin hydrate (6), 8-((3,3-dimethyloxiran-2-yl)methyl)-7-methoxy-2H-chromen-2-one (7), oxypeucedanin hydrate monoacetate (8), xanthotoxin (9), 4-((2-hydroxy-3-methylbut-3-en-1-yl)oxy)-7H-furo[3,2-g]chromen-7-one (10), imperatorin (11) and osthol (12). The isolates were identified using spectral techniques in the light of literature. 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity screening of the isolated constituents was carried out against six human cancer cell lines including lung (A549 and NCI-H322), epidermoid carcinoma (A431), melanoma (A375), prostate (PC-3) and Colon (HCT-116) cell lines. Osthol (12) exhibited the highest cytotoxicity with IC50 values of 3.2, 6.2, 10.9, 14.5, 24.8, and 30.2 µM against epidermoid carcinoma (A431), melanoma (A375), lung (NCI-H322), lung (A549), prostate (PC-3) and colon (HCT-116) cell lines respectively. Epidermoid carcinoma cell line A431 was sensitive to most of the compounds followed by lung (A549) cancer cell line. Finally a simple and reliable HPLC method was developed (RP-HPLC-DAD) and validated for the simultaneous quantification of these cytotoxic constituents in Prangos pabularia. The extract was analyzed using a reversed-phase Agilent ZORBAX eclipse plus column C18 (4.6×250 mm, 5 µm) at 250 nm wavelength using a gradient water-methanol solvent system at a flow rate of 0.8 ml/min. The RP-HPLC method is validated in terms of recovery, linearity, accuracy and precision (intra and inter-day validation). This method, because of shorter analysis time, makes it valuable for the commercial quality control of Prangos pabularia extracts and its future pharmaceutical preparations.

Function of DNA methyltransferase 3a in lead (Pb2+)‐Induced Cyclooxygenase‐2 gene

Lead ions (Pb(2+) ) are toxic industrial pollutants associated with chronic inflammatory diseases in humans and animals. Previously, we found that Pb(2+) ions induce COX-2 gene expression via the EGF receptor/nuclear factor-κB signal transduction pathway in epidermoid carcinoma cell line A431. In this study, to see whether Pb(2+) ions affect COX-2 expression by epigenetic mechanisms, we looked at the mRNAs of DNA methyltransferases (DNMTs) using real-time PCR of total RNA from these cells. Cells exposed to Pb(2+) had low levels of DNMT3a mRNA, whereas the levels of DNMT1 and DNMT3b mRNAs remained unchanged. Pretreatment of cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5 μM) followed by Pb(2+) (1 μM) significantly increased levels of COX-2 mRNA compared with cells treated with Pb(2+) alone. Overexpression of tumor suppressor gene Rb correlated with an increase in COX-2 mRNA and a decrease in DNMT3a mRNA. Conversely, overexpression of transcription factor E2F1 correlated with a decrease in COX-2 mRNA and an increase in DMNT3a mRNA. Pretreatment with EGFR inhibitors AG1478 and PD153035 significantly limited Pb(2+) -induced reduction in DNMT3a mRNA. In addition, gene knockdown of DNMT3a with short hairpin RNA correlated with increased COX-2 mRNA induced by Pb(2+) . Our findings suggest Pb(2+) ions induce COX-2 expression indirectly by reducing DNMT3a methylation of the COX-2 promoter via transcription factors Rb and E2F1.

Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

Cetuximab is a chimeric mouse–human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%–20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab.

Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR

The epidermal growth factor receptor (EGFR), overexpressed in many epithelial tumors, is emerging as an attractive target for cancer therapy. Antibodies to the extracellular region of EGFR play a key role in the development of a mechanistic understanding and cancer therapy. In the present study, we demonstrated for the first time that EGFR-truncated extracellular domain (EGFR-tED), which was expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies, could be purified and renatured. The EGFR-tED protein was purified by gel filtration and Ni-NTA affinity chromatography with high purity (>90%) and refolded by a urea gradient size-exclusion chromatography, which could bind its ligand EGF in a concentration-dependent manner. The renatured EGFR was used for biopanning anti-EGFR scFvs from a human synthetic antibody phage display library. Combined with an additional cell-based ELISA screen, a novel scFv, E10, was obtained with two-fold more potent on the binding to EGFR-bearing tumor cells (the epidermoid carcinoma cell line A431) and the inhibition of A431 cells proliferation than scFv 11F8, suggesting that the E10 has the potential to be developed as therapeutic agents to solid tumors associated with EGFR overexpression.