calcein-am and Lung-Neoplasms

Calcein acetoxymethyl ester (calcein-AM) treatment has been reported to exert antitumor effects in certain cancer cells; however, the detailed mechanism of action of calcein-AM in cancers remains unclear, especially in nonsmall cell lung cancer (NSCLC). This study focused on the function and mechanism of action of calcein-AM in NSCLC. We used cell viability assays, western blotting, and EdU proliferation assay combined with calcein-AM treatment or siRNA interference to investigate the role of topoisomerase IIβ binding protein 1 (TopBP1) and p53 in NSCLC chemotherapy. We found that calcein-AM has antitumor effects in lung cancer and enhances the antitumor effects of doxorubicin in NSCLC. Furthermore, we found that TopBP1, which we previously showed was involved in doxorubicin resistance through upregulation of aberrant p53, was involved in calcein-AM-mediated increased doxorubicin sensitivity. Doxorubicin upregulated the expression of aberrant p53. Calcein-AM repressed the expression of TopBP1, which resulted in reduced expression of aberrant p53 and disrupted the antiapoptotic activity mediated by the TopBP1/mutp53 pathway in NSCLC. Together, our findings show that calcein-AM, the cell-permeable derivative of calcein, exerts significant antitumor effects in NSCLC, and can enhance the antitumor effect of doxorubicin by regulating the TopBP1/mutp53 pathway. These findings provide novel insight into lung cancer treatment.

Topics: A549 Cells; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Line, Tumor; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drug Synergism; Fluoresceins; Humans; Lung Neoplasms; Nuclear Proteins; Signal Transduction; Tumor Suppressor Protein p53

Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.

Topics: Adenocarcinoma; Animals; Biomechanical Phenomena; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Computer Simulation; Endothelial Cells; Endothelium, Vascular; Epithelial Cells; Finite Element Analysis; Fluoresceins; Fluorescent Dyes; Humans; Lung Neoplasms; Mechanotransduction, Cellular; Mice; Myoblasts; Skin; Substrate Specificity

Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, -32, and -43. Connexin26 and -32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and -32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells.

Topics: Cell Communication; Cell Differentiation; Connexin 26; Connexin 43; Connexins; Fluoresceins; Fluorescent Dyes; Gap Junctions; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Microscopy, Fluorescence; Neoplasm Proteins; Nerve Tissue Proteins; Neurons; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured