Human Epidermal Carcinoma Cell Line Research Articles

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3102.

The Desmoglein‐1 Transmembrane Domain Drives Lipid Raft Association and Desmosome Assembly

The epidermis is a barrier to the outside world, but mutations in genes encoding desmosomal proteins can inhibit this function, causing human diseases of skin fragility. Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a severe homozygous recessive disorder of skin fragility caused by mutations in the gene encoding desmoglein 1 (DSG1), a component of desmosomes. Desmosomes are junctional complexes that function in mechanical stress resistance across tissues. In these junctions, desmosomal cadherins such as DSG1 interact across extracellular spaces and with intracellular adaptor proteins to link cytoskeletal elements of adjacent cells. Both assembly and disassembly of desmosomes have been shown to rely on lipid raft membrane microdomains, highly ordered cholesterol and sphingolipid‐enriched regions of the plasma membrane important in signaling and trafficking. Our current model predicts that raft association promotes clustering of desmosomal proteins to drive assembly and mediate adhesion. However, the mechanisms of raft association and desmosome assembly are not fully understood. We created a DSG‐null A431 cell line to examine the relationship between DSG raft association and desmosomal adhesion. A431s are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies that express a single DSG species, DSG2. The absence of DSG2 in these cells was confirmed by western blot and immunofluorescence. Further characterization of these DSG‐null A431 cells revealed mislocalized desmoplakin, a marker of mature desmosomes, and a lack of strong cell‐cell adhesion. Exogenous stable expression of GFP‐tagged wildtype DSG1 (DSG1WT‐GFP) rescued both desmosome formation and function, suggesting that these DSG‐null cells would be suitable for probing the relationship between DSG raft association and desmosome assembly and function. Recently, we discovered a novel, heterozygous mutation in the transmembrane domain (TMD)‐encoding region of DSG1 that causes a glycine to arginine substitution and SAM syndrome. DSG1SAM is a lipid raft targeting mutant suggesting an important role for the TMD in raft association. Stable expression of DSG1SAM‐GFP could not rescue desmosome formation or function in the DSG‐null cells, suggesting that a loss of raft association also impacts desmosome assembly and adhesive function. Intriguingly, a chimeric DSG1 protein containing the TMD from E‐cadherin (DSG1Ecad‐GFP), a non‐raft associating adhesive protein, only partly rescued desmosome formation and function. Furthermore, these results correlated with the predicted lipid raft affinity of each TMD, suggesting that desmosome assembly and function is dependent on raft association. We are currently analyzing a panel of GFP‐tagged DSG1TMD variants with differential raft targeting affinities and assessing their ability to support desmosome assembly and function. In conclusion, the TMD targets desmogleins to lipid rafts and modulates desmoglein adhesive function.Support or Funding InformationNIH R01AR050501, NIH R01AR048266

99m Tc-anti-epidermal growth factor receptor nanobody for tumor imaging.

Nanobodies are important biomolecules for tumor targeting. In this study, we synthesized and labeled anti-epidermal growth factor receptor (EGFR) nanobody OA-cb6 with 99m Tc(CO)3+ and evaluated its characteristics for targeting the EGFR in the A431 human epidermal carcinoma cell line. Nanobody radiolabeling was achieved with high yield and radiochemical purity, and the radioconjugate was stable. Biodistribution results in nude mice exhibited a favorable tumor-to-muscle ratio at 4-hr postinjection, and tumor location was visualized at 4hr after injection of radiolabeled nanobody. Our result showed that the OA-cb6-99m Tc-tricarbonyl radiolabeled nanobody is a promising radiolabeled biomolecule for tumor imaging in cancers with high EGFR overexpression.

A Metal-containing Nucleoside That Possesses Both Therapeutic and Diagnostic Activity against Cancer

Nucleoside transport is an essential process that helps maintain the hyperproliferative state of most cancer cells. As such, it represents an important target for developing diagnostic and therapeutic agents that can effectively detect and treat cancer, respectively. This report describes the development of a metal-containing nucleoside designated Ir(III)-PPY nucleoside that displays both therapeutic and diagnostic properties against the human epidermal carcinoma cell line KB3-1. The cytotoxic effects of Ir(III)-PPY nucleoside are both time- and dose-dependent. Flow cytometry analyses validate that the nucleoside analog causes apoptosis by blocking cell cycle progression at G2/M. Fluorescent microscopy studies show rapid accumulation in the cytoplasm within 4 h. However, more significant accumulation is observed in the nucleus and mitochondria after 24 h. This localization is consistent with the ability of the metal-containing nucleoside to influence cell cycle progression at G2/M. Mitochondrial depletion is also observed after longer incubations (Δt ∼48 h), and this effect may produce additional cytotoxic effects. siRNA knockdown experiments demonstrate that the nucleoside transporter, hENT1, plays a key role in the cellular entry of Ir(III)-PPY nucleoside. Collectively, these data provide evidence for the development of a metal-containing nucleoside that functions as a combined therapeutic and diagnostic agent against cancer.

Combinational Delivery of Hydrophobic and Hydrophilic Anticancer Drugs in Single Nanoemulsions To Treat MDR in Cancer

In this study, we developed the core-matched nanoemulsions (NEs) functionalized by vitamin E (VE) and tocopherol poly(ethylene glycol)succinate (TPGS) to codeliver hydrophobic and hydrophilic drugs, paclitaxel (PTX) and 5-fluoroucacil (5-FU), in order to achieve synergistic effects and overcome PTX resistance in a multi-drug-resistant (MDR) human epidermal carcinoma cell line KB-8-5. Antitumor effect of the combination therapy based on core-matched technology (CMT) was evaluated in vitro and in vivo in mice. The core-matched NEs showed entrapment efficiency of >90% and were of nanoscale particle size and negative zeta-potential. The combined core-matched NEs exhibited concentration and time-dependent cytotoxicity against PTX-sensitive KB-3-1 cells and PTX-resistant KB-8-5 cells as well as an obviously increased G2/M phase block. The improvements in therapeutic response over either PTX–VE or 5-FU–TPGS therapy alone were demonstrated by the ability to effectively induce the apoptosis of tumor cells via up-regulation of tumor suppressor p53 and β-tubulin and by the significant inhibition of cell cycle progression. The combination therapy led to dramatic inhibition of tumor growth with little toxicity in vivo, especially in the PTX-resistant KB-8-5 tumors, whereas Taxol had little therapeutic effect. This was mainly ascribed to the synergism of PTX and 5-FU and the reverse of MDR by the inhibition of ATPase activity by VE and TPGS. Coencapsulation of two chemotherapeutic agents with different mechanisms allows simultaneous interruption of diverse anticancer pathways, resulting in increased therapeutic response and low toxicity. The CMT markedly facilitated the long circulation of PTX and 5-FU, which was closely associated with the high accumulation of chemotherapeutic agents within the tumors and the improvement of antitumor efficacy. The current study demonstrated the feasibility of incorporating PTX and 5-FU targeting to different pathways into a single core-matched NE for the reversal of MDR and synergism in cancer therapy.

Metalloprobes: Synthesis, characterization, and potency of a novel gallium(III) complex in human epidermal carcinoma cells

Multidrug resistance (MDR) mediated by overexpression of the MDR1 gene product, P-glycoprotein (Pgp), represents one of the best characterized barriers to chemotherapeutic treatment in cancer and may be a pivotal factor in progression of Alzheimer’s disease (AD). Thus, agents capable of probing Pgp-mediated transport could be beneficial in biomedical imaging. Herein, we synthesized and structurally characterized a gallium(III) complex ( 5) of the naphthol–Schiff base ligand. The crystal structure revealed octahedral geometry for the metallodrug. Cytotoxicity profiles of 5 were evaluated in KB-3-1 (Pgp−) and KB-8-5 (Pgp+) human epidermal carcinoma cell lines. Compared with an LC 50 (the half-maximal cytotoxic concentration) value of 1.93 μM in drug-sensitive (Pgp−) cells, the gallium(III) complex 5 demonstrated an LC 50 value > 100 μM in drug-resistant (Pgp+) cells, thus indicating that 5 was recognized by the Pgp as its substrate, thereby extruded from the cells and sequestered away from their cytotoxic targets. Radiolabeled analogues of 5 could be beneficial in noninvasive imaging of Pgp-mediated transport in vivo.

Augmentation of chemotherapy-induced cytokine production by expression of the platelet-activating factor receptor in a human epithelial carcinoma cell line.

In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-alpha induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-kappaB were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NFkappaB pathway using a super-repressor IkappaBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-kappaB-dependent process.

The Epidermal Platelet-activating Factor Receptor Augments Chemotherapy-induced Apoptosis in Human Carcinoma Cell Lines

Most chemotherapeutic agents exert their cytotoxic effects in part through the induction of apoptosis. In addition, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors, it is not known whether PAF is involved in chemotherapeutic agent-induced apoptosis. These studies examined the role of the PAF system in chemotherapy-mediated cytotoxicity using model systems created by retroviral mediated transduction of the PAF receptor-negative human epidermal carcinoma cell line KB with the human PAF receptor (PAF-R) and ablation of the endogenous PAF-R in the carcinoma cell line HaCaT with a retroviral mediated inducible antisense PAF-R vector. The presence of the PAF-R in these models resulted in an augmentation of apoptosis induced by chemotherapeutic agents etoposide and mitomycin C but not by tumor necrosis factor-related apoptosis-inducing ligand or by C(2) ceramide. Oxidative stress and the transcription factor nuclear factor kappaB (NF-kappaB) are found to be involved in this augmentative effect because it was blocked by antioxidants and inhibition of the NF-kappaB pathway using a super-repressor form of inhibitor B. These studies provide evidence for a novel pathway whereby the epidermal PAF-R can augment chemotherapy-induced apoptotic effects through an NF-kappaB-dependent process.

Stress-activated Protein Kinase/Jun N-terminal Kinase Is Required for Interleukin (IL)-1-induced IL-6 and IL-8 Gene Expression in the Human Epidermal Carcinoma Cell Line KB

The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKbeta (SAPKbeta(K-R)) or antisense RNA to SAPKbeta in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFkappaB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKbeta(K-R), and three clones stably overexpressing antisense RNA to SAPKbeta, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.

Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-α on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils

The present study was undertaken to determine the identities and characteristics of proteins with molecular masses between 40 and 44 kDa whose tyrosine phosphorylation increases in human neutrophils following stimulation of these cells with tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunoblotting results demonstrate that addition of GM-CSF to human neutrophils increases the tyrosine phosphorylation of two proteins with molecular masses of 42 and 44 kDa. However, the addition of TNF-alpha to neutrophils induces a time- and dose-dependent increase in tyrosine phosphorylation of a 40 kDa protein. Immunoprecipitation using specific mitogen-activated protein kinase (MAPK) isoform antibodies and an antibody which recognizes phosphotyrosine-containing proteins demonstrated that the 42 and 44 kDa proteins are isoforms of MAPKs. Utilizing an in situ gel kinase activity assay, GM-CSF increases the kinase activity of the 42 and 44 kDa proteins. Moreover, using immunoprecipitated p42 and p44 MAPK isoforms in this gel assay revealed activity associated with the p42 and p44 MAPK isoforms. Using the same in situ assay, TNF-alpha induces an increase in kinase activity of a 40-42 kDa protein. However, the 40 kDa protein whose phosphorylation on tyrosine residues increased in human neutrophils following stimulation with TNF-alpha is not a member of the known MAPK family, demonstrating the divergences in pathways utilized by GM-CSF and TNF-alpha. This 40 kDa protein may be related to the recently identified protein that becomes phosphorylated on tyrosine residues upon stimulation of the human epidermal carcinoma cell line KB by interleukin-1. In these cells the p40 protein is part of a protein kinase cascade which results in the phosphorylation of the small heat shock protein, hsp27.

Purification and characterization of a cytosolic transglutaminase from a cultured human tumour-cell line.

Transglutaminases are a family of Ca2(+)-dependent enzymes that catalyse the formation of isopeptide bonds between the side chains of glutamine and lysine residues. The enzymes have been hypothesized to be involved in a wide range of cellular processes, including growth and differentiation and stabilization of the cytoskeleton. The human epidermal carcinoma-cell line, A431 cells, have relatively high amounts of a cytosolic transglutaminase activity that varies upon treatment of the cells with epidermal growth factor. We demonstrate here that this cytosolic activity has the biochemical and immunological properties of a tissue transglutaminase. We also report the purification of this enzyme to apparent homogeneity by a protocol which involves a novel affinity-elution step. Polyclonal antibodies to the transglutaminase were raised and used to identify the enzyme by Western blotting. The availability of purified transglutaminase and antitransglutaminase antibodies will permit further study of the role of this enzyme in the growth of this hormone-responsive human tumour-cell line.

Rapid increases in the transglutaminase activity of A431 cells following treatment with epidermal growth factor.

Transglutaminase activity was detected in lysates of A431 cells, a human epidermal carcinoma cell line. Enzyme activity was increased 1.5-2.5-fold in lysates prepared from cells pretreated with epidermal growth factor (EGF) relative to untreated control cells. Half-maximal activation of the transglutaminase activity occurred at 3-5 nM EGF, a concentration in good agreement with the Kd for EGF binding to its receptor in these cells. The increase in transglutaminase activity could be detected as early as 2 min after the addition of EGF, with the maximal response attained by 30 min. The activation was not blocked by pretreatment of the cells with cycloheximide, suggesting that the increased activity was not the result of an induction of transglutaminase synthesis. Fractionation of A431 cell lysates by centrifugation at 100000g for 30 min demonstrated that 90% of the transglutaminase activity was present in the soluble fraction and that this soluble transglutaminase activity was increased after treatment of the cells with EGF. The demonstration that EGF acutely increases the activity of a soluble, intracellular transglutaminase defines a novel pathway of growth factor action and provides a useful model system for identifying and comparing the mechanism(s) by which growth factors activate soluble enzymes.

Effects of Alpha and Beta Interferons on Cultured Human Keratinocytes

Interferons (IF) are a family of glycoproteins known for their antiviral activity and the ability to inhibit growth and alter behavior of various normal and transformed cell types, both in vivo and in vitro. Because cultured human keratinocytes (HK) produce IF in response to viral infection, we undertook studies of alpha-IF and beta-IF effects on HK. Cloned human IF were added at time of seeding and at each feeding to paired dishes of keratinocytes maintained in serum-free hormone-supplemented medium. At 7 days significant inhibition of growth was observed for both alpha-IF and beta-IF, as determined by cell counts, total protein, and appearance of stained colonies, and was sustained for at least two weeks during continuous IF exposure. The inhibition was substantially blocked by prior addition of cholera toxin to the medium, consistent with competition for a common cell surface receptor. Growth of a single human epidermal carcinoma cell line was much less affected by IF than was growth of the normal keratinocytes. IF also promoted terminal differentiation of keratinocytes as assessed by desquamation rate of cells from the colony surface and by proportion of total cells having cornified envelopes. IF effect on both growth and differentiation was completely reversible within days of its removal from medium. These findings suggest that IF may function as a physiologic regulator of epidermal growth in vivo with properties of a negative growth factor or chalone.

Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin.

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.