Human Epidermoid Carcinoma A431 Research Articles

ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

Abstract 6720: HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors

Abstract Introduction/Purpose: The Wagner lab has developed a protein nanoring based platform to direct human immune cells to combat solid tumors. These nanorings are bispecific, comprised of tumor-targeting and immune cell-targeting dihydrofolate reductase (DHFR2) fusion proteins. These protein nanorings direct immune cells to target and destroy cancer cells. For treating solid tumors, we aimed to target epidermal growth factor receptor (EGFR). However, targeting EGFR could lead to potential “on-target, off-tumor” effects due to expression of EGFR in healthy tissue. Matrix metalloproteinases are overexpressed in a variety of solid tumors. Therefore, to improve tumor specificity, we hypothesized that masking the immune cell targeting DHFR2 protein through a matrix metalloproteinase-2 (MMP-2) sensitive linker will prevent engagement and activation of immune cells outside the tumor microenvironment (TME). Once these protease-activatable nanorings enter the TME and bind to EGFR, cleavage by MMP-2 will lead to unmasking of the immune cell binder, thereby crosslinking and activating immune cells, causing targeted cancer cell lysis. Methods: We designed human histidine triad nucleotide binding protein-1 (HINT1) masked anti-immune cell targeting nanobody-DHFR2 fusion proteins. HINT1 was hypothesized to sterically block the engagement of immune cells outside the tumor. HINT1-anti-TCR-DHFR2 (TCR = T cell receptor) and HINT1-anti-CD16-DHFR2 were cloned, expressed, purified, and characterized. Their apparent binding affinities were determined using flow cytometry. The anti-tumor efficacy of these nanorings was evaluated against EGFR overexpressing A431 (human epidermoid carcinoma) cells. Results: HINT1-anti-TCR-DHFR2 and HINT1-anti-CD16-DHFR2 rings showed reduced binding to T and NK-92-CD16 cells, respectively. In vitro MMP-2 cleavage assay showed complete removal of the HINT1 mask within 2 hours. Western blot analysis showed that both proteins were stable in human serum up to 3 days. 2D cytotoxicity assays with protein nanorings assembled using anti-EGFR-DHFR2 and anti-TCR-DHFR2 or anti-CD16-DHFR2 with T and NK-92-CD16 cells respectively, showed a potent lysis of A431 cells. The EGFR-targeted lysis was diminished in the presence of HINT1 mask, thereby providing proof-of-concept for the steric masking of the anti-immune cell targeting nanobodies. Conclusion: Protease-activatable nanorings can selectively lyse EGFR+ tumor cells in vitro. Their antitumor efficacy and safety are currently being evaluated using a previously established in vivo mouse model. Therefore, HINT1-steric masking can serve as a simple and effective strategy to address off-tumor toxicities associated with bispecific immunotherapies. Citation Format: Abhishek Kulkarni, Debasmita Paul, Declan Dahlberg, Kostana Ligori, Freddys Rodriguez, Lakmal Rozumalski, Bruce Walcheck, Carston R. Wagner. HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors [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 6720.

TARGET ONCOGENIC RECEPTORS IN TUMOURS, FROM ITS INITIAL CLINICAL BREAKTHROUGHS TO CURRENT CLINICAL STANDARD THERAPY

Epidermal growth factor (EGF) is a polypeptide hormone originally isolated from mouse submaxillary gland, which is a potent mitogen for a wide variety of culture cells of both epithelial and mesenchymal origin, and its important role in the proliferation, differentiation, and survival of neural and glial precursor cells. The physiological effects of EGF are mediated by an EGF receptor with tyrosine-specific protein kinase activity. The traditionally accepted view is that normal epidermal growth factor receptor (EGFR) is no tumorigenic, whereas mutated EGFR such as an oncogenic receptor EGFRvIII is oncogenic. In recent, it has been implicated that EGF could be beneficial for burn, wound healing, diabetic foot ulcer, and show an attractive perspective. In addition, cosmetic containing EGF would be effective in improving the plasticity of skin, helps in anti-aging and whitening, and controls the amount of erythema and sebum in the skin. In our team we have successfully prepared a series of 350 bottles of Shampoo liquid and 26 bottles of recombinant human EGF (rhEGF) spray, and 4 bottles of EGF-Silvadence ointment. The initial results indicated that prepared rhEGF is safe and available in clinical use. On the other hand, progress on the interaction of EGF with its altered oncogenic receptor signaling through its downstream molecules such Ras/Raf/MAPK (see figure in the full text, George Zhu,1991) and/or PI3k/akt pathway in the development of some cancers such as A431 human epidermoid carcinoma cells, brain glioblastoma, breast, pancreas and lung cancers. In addition to a series of gefitinib, erlotinib, osimertinib and the CIMA vax-EGF vaccine, an antioncogenic receptor antibody based fusion protein [for example Cetuximab-based IL-10 fusion protein, CmAb-(IL10)2] provide a potential strategy to improve cancer immunotherapy. Peer Review History: Received 1 December 2023; Revised 26 January 2024; Accepted 2 March; Available online 15 March 2024 Academic Editor: Dr. Tamer Elhabibi, Suez Canal University, Egypt, tamer_hassan@pharm.suez.edu.eg Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 8.0/10 Reviewers: Dr. Bilge Ahsen KARA, Ankara Gazi Mustafa Kemal Hospital, Turkey, ahsndkyc@gmail.com Dr. Asia Selman Abdullah, University of Basrah, Iraq, asia_abdullah65@yahoo.com

Phytochemical characterization and biological activities of Stenomesson miniatum bulb extract, a medicinal plant of the Andes

Fresh bulbs of Stenomesson miniatum, a plant belonging to the Amaryllidaceae family with a poorly investigated phytochemical profile, were traditionally employed by Andean healers to treat tumors and abscesses. The aims of this study were to characterize the extract from the bulbs of S. miniatum and to test its cytotoxic and antibacterial potential. A previous structural study of the major extract constituents was extended to include the minor components by means of 13C-NMR-based dereplication. Cytotoxic activities were evaluated on A431 human epidermoid carcinoma cells through a metabolic assay and on Jurkat human acute T-leukemia cells through a cell-impermeant fluorescent nuclear probe. Antibacterial assays were carried out against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes by using a standardized broth microdilution method. Eleven known Amaryllidaceae alkaloids were identified together with another compound determined as being an extraction artefact. The alkaloid-enriched extract showed good cytotoxic activity against both tumor cell lines, reaching an IC50 of 3.3 µg/mL against A431 cells and of 10.9 µg/mL against Jurkat cells. Biological assays carried out on single fractions showed that activity can be attributed to the presence of pretazettine and haemanthamine. Conversely, no antibacterial activities were recorded for any of the samples.

Prospective in vitro A431 cell line anticancer efficacy of zirconia nanoflakes derived from Enicostemma littorale aqueous extract.

Biomedical applications of zirconia nanomaterials were limited in biological systems. In this research, 8-15 nm size zirconia nanoflakes (ZrNFs) were fabricated and their nature, morphology, and biocompatibility were evaluated. The synthesis was carried out using Enicostemma littorale plant extract as an effective reducing and capping agent. Physiochemical properties of prepared ZrNFs were characterized using diverse instrumental studies such as UV-vis spectrophotometer, Fourier-transform infrared, powder X-ray diffractometer, scanning electron microscope, transmission electron microscope (TEM), energy dispersive X-ray, and cyclic voltammetry (CV). The XRD pattern confirmed the tetragonal phases of ZrNFs and the highest crystallite size of Zr0.02, Zr0.02, and Zr0.06 was 56, 50, and 44 nm, respectively. The morphology of samples was assessed using TEM. Electrophysiological effects of ZrNFs in the cellular interaction process were revealed by the slower rate of electron transfer results in CV demonstration. Biocompatibility of synthesized ZrNFs was studied on A431 human epidermoid carcinoma epithelial cells. The cell viability was increased with an increasing the concentration of nanoflakes up to 6.50-100 μg/mL. The cell viability and observed IC50 values (44.25, 36.49, and 39.62 μg/mL) reveals that the synthesized ZrNFs using E. littorale extract is found to be efficient toxic to A431 cancer cell lines.

Synthesis and in vitro photodynamic activity of aza-BODIPY-based photosensitizers.

Aza-BODIPY dyes have recently come to attention owing to their excellent chemical and photophysical properties. In particular, their absorption and emission maxima can efficiently be shifted to the red or even to the NIR spectral region. On this basis, aza-BODIPY derivatives are widely investigated as fluorescent probes or phototherapeutic agents. Here we report the synthesis of a set of novel aza-BODIPY derivatives as potential photosensitizers for use in photodynamic therapy. Triazolyl derivatives were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition as the key step. In vitro photodynamic activities of the newly synthesized compounds were evaluated on the A431 human epidermoid carcinoma cell line. Structural differences influenced the light-induced toxicity of the test compounds markedly. Compared to the initial tetraphenyl aza-BODIPY derivative, the compound bearing two hydrophilic triethylene glycol side chains showed substantial, more than 250-fold, photodynamic activity with no dark toxicity. Our newly synthesized aza-BODIPY derivative, acting in the nanomolar range, might serve as a promising candidate for the design of more active and selective photosensitizers.

In Situ Hybridization (ISH) Combined with Immunocytochemistry (ICC) Co-detection of Phosphorylated EGFR in A431 Cultured Cells.

Antibodies have been commonly used to study protein phosphorylation since the first phospho-specific antibody was described in 1981. Antibodies can be developed so that they specifically recognize phosphorylated areas of particular proteins. In situ hybridization (ISH) is the technique where specific RNA or DNA molecules can be detected in a single cell without the need for antibodies. Using ACD's integrated Co-Detection Workflow (ICW), we have developed a protocol to use phospho-specific antibodies in combination with ISH to show co-localization of EGFR mRNA and EGFR proteins phosphorylated at different sites in tumor cells. Our protocol has been used for multiplexing Y1086 phosphorylated EGFR, Y1068 phosphorylated EGFR, and EGFR RNA in A431 human epidermoid carcinoma cells.

Preparation of radiolabeled erlotinib analogues and analysis of the effect of linkers

Erlotinib is a first generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) which was granted Food and Drug administration (FDA) approval for treatment of patients with locally advanced or metastatic NSCLC. The present study aimed at development of radiolabeled erlotinib variants as tyrosine kinase inhibitors. Three DOTA-erlotinib conjugates were prepared for radiolabeling with 177Lu. The terminal alkyne group of erlotinib was modified by performing Cu-catalyzed click chemistry and three different linkers were introduced which were then conjugated to the chelator, DOTA. The DOTA-erlotinib conjugates were characterized by 1H NMR and ESI-MS. 177Lu-DOTA-erlotinib complexes were characterized using natLu-DOTA-erlotinib conjugates. The 177Lu-complexes exhibited high in vitro stability in human serum up to 48 h. They were highly hydrophilic in nature as observed from their log Po/w values (177Lu-DOTA-propyl-Er: −2.5 ± 0.1; 177Lu-DOTA-PEG3-Er: −3.0 ± 0.1; 177Lu-DOTA-PEG6-Er: −3.3 ± 0.1). The MTT assay in A431 human epidermoid carcinoma cell lines indicates that the chemical modification at the terminal alkyne group of the erlotinib molecule does not have significant effect on its TKI property. Biodistribution studies in normal Swiss mice demonstrated fast clearance and excretion of 177Lu-labeled erlotinib complexes. These studies indicate that erlotinib variants with hydrophobic pharmacokinetic modifiers/chelators may enhance the retention of 177Lu-labeled complexes in blood thereby increasing the probability to reach EGFR-expressing tumor.

Investigation of 3-sulfamoyl coumarins against cancer-related IX and XII isoforms of human carbonic anhydrase as well as cancer cells leads to the discovery of 2-oxo-2H-benzo[h]chromene-3-sulfonamide - A new caspase-activating proapoptotic agent.

Herein we report the synthesis of a set of seventeen 3-sulfonamide substituted coumarin derivatives. Prepared compounds were tested invitro for inhibition of four physiologically relevant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Several coumarin sulfonamides displayed low nanomolar KI values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Some of these compounds exerted a concentration-dependent antiproliferative action toward RT4 human bladder cancer and especially A431 human epidermoid carcinoma cell lines. In the meantime, the viability of non-tumorigenic hTERT immortalized human foreskin fibroblast cell line Bj-5ta was not significantly affected by the obtained derivatives. Interestingly, compound 10q (2-oxo-2H-benzo [h]chromene-3-sulfonamide) showed a profound and selective dose-dependent inhibition of A431cell growth with low nanomolar IC50 values. We demonstrated that 10q possessed a concentration-dependent apoptosis induction activity associated with caspase 3/7 activation in cancer cells. As carbonic anhydrase isoforms in question were not potently inhibited by this compound, its antiproliferative effects likely involve other mechanisms, such as DNA intercalation. Compound 10q clearly represents a viable lead for further development of new-generation anticancer agents.

Natural Compounds Isolated from Stachybotrys chartarum Are Potent Inhibitors of Human Protein Kinase CK2

A large number of secondary metabolites have been isolated from the filamentous fungus Stachybotrys chartarum and have been described before. Fourteen of these natural compounds were evaluated in vitro in the present study for their inhibitory activity towards the cancer target CK2. Among these compounds, stachybotrychromene C, stachybotrydial acetate and acetoxystachybotrydial acetate turned out to be potent inhibitors with IC50 values of 0.32 µM, 0.69 µM and 1.86 µM, respectively. The effects of these three compounds on cell proliferation, growth and viability of MCF7 cells, representing human breast adenocarcinoma as well as A427 (human lung carcinoma) and A431 (human epidermoid carcinoma) cells, were tested using EdU assay, IncuCyte® live-cell imaging and MTT assay. The most active compound in inhibiting MCF7 cell proliferation was acetoxystachybotrydial acetate with an EC50 value of 0.39 µM. In addition, acetoxystachybotrydial acetate turned out to inhibit the growth of all three cell lines completely at a concentration of 1 µM. In contrast, cell viability was impaired only moderately, to 37%, 14% and 23% in MCF7, A427 and A431 cells, respectively.

Abstract LB187: EGFR signaling and pharmacology in oncology revealed with an innovative RTK biosensor technology

Abstract EGFR is involved in key biological processes and its deregulation is associated with the development of many cancers. EGFR is implicated in tumor invasion, metastasis and angiogenesis. Moreover, intrinsic and acquired mutations of EGFR have been described to modify receptor signaling and be responsible for the appearance of drug resistance during treatment in a clinical setting. Development of tools providing new insight into RTK complex mechanisms is crucial to develop more effective RTK-targeting drugs. In this study, we present a live-cell ebBRET-based biosensor platform which includes 12 distinct biosensors for monitoring SH2-domain containing proteins-mediated downstream signaling of RTKs, hence following the activation of MAPK, Akt and PKC pathways. These biosensors are designed to measure receptor proximal events that are engaged upon receptor activation at the plasma membrane (PM) and early endosomal compartments (EE). Using EGFR and two of its ligands as a model system, we showed the capacity of these biosensors to differentiate unique signaling signatures of EGFR, at the PM or the EE, with EGF and Epiregulin ligands displaying differences of efficacy and potency. Indeed, EGF was more potent and efficacious than Epiregulin on all the studied pathways. Also, we detected activities at the EE with EGF stimulation but not with Epiregulin. Our data highlight the platform's capacity to follow the trafficking of RTK biosensors into different compartments and to reveal internalization selectivity and bias which can be observed with different ligands. Overcoming resistance has become an important challenge when developing new therapeutics for the treatment of cancer. We further demonstrated that EGFR deletions or single point mutations, found in Gliobastoma or NSCLC, have an impact on the constitutive activity of EGFR and the signaling profiles but also on inhibitor efficacies which could impact receptor trafficking from the PM to the EE. In addition, the sensitivity of the RTK platform allowed us to measure the signaling of endogenously expressed EGFR in pathophysiologically-relevant cell lines commonly used for their oncogenic properties, such as A431 human epidermoid carcinoma cells, N87 gastric carcinoma cells and T47D, MCF7 and SK-BR-3 breast adenocarcinoma cells. Finally, we illustrated, using BRET-based imaging, the recruitment of SH2 effectors at the PM or at the EE after a 10-minute or 60-minute incubation respectively with EGF, highlighting the translatability of the biosensor platform to microscopy. The ebBRET-based biosensor technology displayed new insights in RTK biology and revealed different modes of action, extensive RTK signal profiling, and trafficking of RTK effectors. It represents a powerful tool for the analysis of RTK mutations and for the identification of novel generation of TKIs and antibodies directed against RTKs. Citation Format: Florence Gross, Guilhem Dugast, Arturo Mancini, Hiroyuki Kobayashi, Michel Bouvier, Stephan Schann, Xavier Leroy, Laurent Sabbagh. EGFR signaling and pharmacology in oncology revealed with an innovative RTK biosensor technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB187.

Synthesis, Cytotoxic Activity and Molecular Docking Studies of New Condensed Thieno[2,3-d]Pyrimidines as Antitumor Agents

A series of twenty molecules based on thienopyrimidine nucleus fused with 5-, 6-, and 7-membered lactam rings as well as triazole were synthesized via 4-step reactions. The target compounds were obtained in good yield and confirmed for their structural integrity. The cytotoxic activity against A431 human epidermoid carcinoma and H9c2 rat cardiomyocyte cells were studied by MTT assay. Most of the synthesized compounds except 8a showed 85% cell death in both human A341 and rat H9c2 cell lines, indicating potential anticancer activity of these compounds. Interestingly, compound 8a showed a reversal in trend by enhancing the growth of both A341 and H9c2 cells.

Pegylated carbon nitride nanosheets for enhanced reactive oxygen species generation and photodynamic therapy under hypoxic conditions

The application of photodynamic therapy (PDT) is of ever-increasing importance in the treatment of malignant tumors; however, there are several major constraints that make it impossible to achieve optimal therapeutic effects. Our objective is to develop a novel photosensitizing drug for skin cancer. In the experiment, we fabricated four-arm-poly ethylene glycol modified amino-rich graphite phase carbon nitride nanosheets (AGCN-PEG), which have good stability in physiological solution and show selective accumulation in tumor cells. Under hypoxic conditions, the AGCN-PEG induced PDT can effectively inhibit growth on A431 human epidermoid carcinoma cells in vivo and in vitro. What's more, after being combined with TMPyP4, the therapeutic effect of AGCN-PEG was greatly improved.

The efficacy of lyticase and \u03b2-glucosidase enzymes on biofilm degradation of Pseudomonas aeruginosa strains with different gene profiles

BackgroundPseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and β-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated.ResultsThis study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa. In contrast to the lyticase enzyme, β-glucosidase reduced the ceftazidime MBECs significantly (P < 0.05). Both enzymes had no cytotoxic effect on the A-549 human lung carcinoma epithelial cell lines and A-431 human epidermoid carcinoma cell lines.ConclusionConsidering the characteristics of the β-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed.

Morphofunctional Differences of Micronuclei in Cultures of Human p53-Positive and p53-Negative Tumor Cells.

Micronuclei that are often present in tumor cells are not only the indicator of genetic instability, they also can induce DNA damage during cell progression through the cell cycle. p53 protein is the key regulator of the cell cycle. In this study we compared morphofunctional features of micronuclei depending on the presence of wild-type p53 gene: in human breast adenocarcinoma MCF-7 (p53+) and human epidermoid carcinoma A431 (p53-). The number of cells with MN in these cell lines does not depend on the presence of active p53. However, micronuclei in cell culture with mutant p53 protein more often have lamina defects, carry DNA damage, and generally determine higher risk of tumor progression. Asynchronous with the main nucleus DNA replication in micronuclei in p53+ and p53- cell cultures demonstrates predisposition of cells with micronuclei to chromothripsis.

Iridium-Containing Polymers Based on Norbornene and 7-Oxa-norbornene Monomers: Synthesis and Photophysical and Biological Properties

Polymers containing oligoether groups, amino acid fragments, and luminophore complexes of iridium(III) in side chains are synthesized by metathesis polymerization. The photophysical properties of the compounds obtained are studied. The iridium-containing copolymers show intense green, blue-green, and red photoluminescence, and the color is determined by the nature of the iridium(III) complexes contained in polymeric emitters. Polymeric products are soluble in water and form micelles with average sizes of 19–54 nm. The cytotoxicity of the polymers with respect to A431 human epidermoid carcinoma cells is determined.

In-vitro Anti-proliferative Effects of Ethanolic Extract of Vanilla Planifolia Leaf Extract against A431 Human Epidermoid Carcinoma Cells

Squamous cell carcinoma is the second largest among skin cancer diseases. The aim of the present study is to reveal the antiproliferative property of vanilla leaf extract against A431 cells. Antiproliferative property was assessed by MTT assay. Apoptotic property was assessed by DNA fragmentation assay. Antiproliferative property of extract was revealed in a dose dependent manner. IC50 of the extract against A431 cells was revealed at 31.2µg/ml. This study revealed the cancer suppression capability of vanilla leaf extract in skin cancer cell lines.

Silibinin induced apoptosis of human epidermal cancer A431 cells by promoting mitochondrial NOS

The antitumor effects of silibinin are of increasing interest, though its mechanism is not yet clear. The goal of this study was to clarify the mechanism of silibinin-induced cell death in the A431 human epidermoid carcinoma cell line. We used a cell viability assay, flow cytometry, nitric oxide (NO) assay, and western blotting to examine relationships between silibinin, NO generation and apoptosis in A431 cells. Silibinin inhibited A431 cell growth in a dose-dependent manner, inducing mitochondrial damage, and apoptosis at a high dose. At the same time, high dose silibinin increased NO levels in A431 cells and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methylester (L-NAME) attenuated silibinin-induced cell growth inhibition. By western blotting, silibinin caused increased eNOS phosphorylation in the mitochondria. The AMP-activated protein kinase inhibitor compound C significantly decreased p-eNOS expression, while blocking eNOS did not affect p-AMPK levels, suggested that AMPK acted upstream of eNOS. This study showed that silibinin increased NO levels in A431 cells by activating the AMPK–eNOS pathway, leading to mitochondrial dysfunction and apoptosis. In this mechanism of action, mitochondrial eNOS played an important role. The results provided new understanding of the functions of intracellular NO.

Cholecystokinin-2 Receptor Targeting with Novel C-terminally Stabilized HYNIC-Minigastrin Analogs Radiolabeled with Technetium-99m.

The high overexpression of cholecystokinin-2 receptors (CCK2R) in tumors, such as medullary thyroid carcinoma, allows for highly specific diagnostic and therapeutic targeting with radiolabeled peptide probes derived from natural ligands for the receptor. Based on the ideal imaging characteristics, high availability and low cost of technetium-99m (99mTc)-labeled radiopharmaceuticals we have developed two hydrazinonicotinic acid (HYNIC) conjugated minigastrin analogs allowing labeling at high specific activity. The CCK2R targeting peptide conjugates show specific amino acid substitutions in the C-terminal receptor-specific sequence with the aim to increase stability and tumor targeting. The CCK2R affinity and the cell uptake of the new radioligands were analyzed using A431 human epidermoid carcinoma cells stably transfected with human CCK2R and mock transfected cells. Metabolic studies in BALB/c mice revealed a high resistance against enzymatic degradation for both radioligands. Biodistribution studies in tumor-xenografted athymic BALB/c nude mice at 1 h and 4 h p.i. showed that the two 99mTc-labeled compounds showed varying uptake in receptor expressing organs, stomach and pancreas (1.3–10.4% IA/g), as well as kidneys, the main route of excretion (7.8–19.9% IA/g). The tumor uptake in A431-CCK2R xenografts was 24.75 ± 4.38% IA/g for [99mTc]Tc-HYNIC-MGS5 and 42.48 ± 6.99% IA/g for [99mTc]Tc-HYNIC-MGS11 at 4 h p.i., whereas the tumor-to-kidney ratio was comparable (2.6–3.3). On demand availability and potential application for radioguided surgery of a 99mTc-labeled minigastrin analog support the further evaluation of these highly promising new compounds.

Entosis and Cell Cycle in Tumor Cell Culture

Entosis is a type of cell cannibalism during which one tumor cell invades another. The fate of the inner cell can vary. It can leave the entotic vacuole, divide within it, or be subjected to lysosome-mediated degradation. The aim of our work was to determine whether MCF7 (p53+) human breast adenocarcinoma cells and A431 (p53-) human epidermoid carcinoma cells can pass through the cell cycle during entosis. The percentage of entotic cells was 1.01 ± 0.37% in MCF7 culture and 0.42 ± 0.27% in A431 culture. It was shown that inner cells, as well as outer cells, can synthesize DNA (BrdU incorporation) and enter mitosis. Morphometric analysis showed polyploidization of outer cells. This process is most pronounced in the A431 (p53–) cell line. In addition, polyploid cells may be the preferred targets of invasion in this culture. In the MCF-7 cell line, the number of G1-phase entotic cells was higher, probably due to p53-mediated cell cycle arrest or the preferred cell invasion in the G1-phase. Overall, in tumors with active p53 protein expression, the entosis contribution to polyploidy and genetic instability of tumor cells is less than in p53 tumor cells.