Nicotine, the addictive component of cigarettes, induces tumor progression via the α7-nicotine acetylcholine receptor (α7-nAChR) subtype in lung cancer. However, the molecular mechanisms by which nicotine impacts tumor progression of breast cancer (BC) remain unclear. In this study, we evaluated the effects of nicotine/smoking on tumor angiogenesis, and epithelial-mesenchymal transition (EMT) signaling mechanisms that are involved in BC progression. BC cell spheroids from the MCF7, a non-invasive cell line was treated with nicotine (0.3 to 3μM) for 14 days. Sections of tumor tissue from the human BC (smoking vs. never smoking) were assessed for phenotypic expressions of α7-nAChR, and vimentin by immunostaining (IHC). CD31 staining was used to evaluate tumor angiogenesis, as measured by microvessel density (MVD). Western blot (WB) analyses were also performed on lysates of both cell lines and tumors to assess the effect of nicotine on the signaling pathways that promote BC progression. The present study revealed that nicotine treated spheroids have increased expression of Ki67, Mdr1, Oct4 signatures that are indicative of BC progression. IHC analysis indicated that tumor from patients that smoked displayed increased expression of α7-nAChR. Furthermore, the expression of Oct4 (marker for cancer stem-cell), and vimentin (marker for EMT) that correlates with advanced disease and metastasis were found to be higher in smokers. However, tumors from non-smokers had low expression of Oct4 and vimentin. It was also found that tumors from patients who smoked had well established vascularity, while tumors from non-smokers had very little vascularization. Indeed, the mean MVD was lower in tumors from non-smokers compared to tumors from smokers. These findings offer significant novel insights into the mechanism by which nicotine contributes to the increased aggressiveness of BC. This work was supported in part by NIH/NCI Workforce Diversity Grant R21-CA171251. Venkat Katkoori, Shreeya R. Kandukuri, Madhukar V. Burra, James E. Trosko, Deimante M. Tamkus, Cherly Leece, Lakshimipathi R. Kareti, George S. Abela, Harvey L. Bumpers. Smoking promotes angiogenesis and epithelial to mesenchymal transition in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3848.

After delivering my Keynote address to the young scientists at the International Gap Junction meeting, “International Colloquium on Gap Junctions and Cancer: Discussing how cellular communication modifies carcinogenesis and cancer treatment outcomes” (San Paulo, Brazil, 10–14 July 2023), I was asked to condense the talk into a manuscript covering the 50+ years of my scientific journey of basic biomedical research. While this journey had to omit so many important experiences, people and external events, I have tried to illustrate how my initial motivation, personal perspective of my abilities, and naïve goal as a young scientist did not prepare me for the non-linear path to where I ended. That path had many twists, turns, barriers, disappointments, surprises and wrong directions, as well as associations with co-workers, students and collaborators who afforded me with great wisdom, support and technical advice. In the history of science, few individuals have left their footprints in a straight path; however, my early education in human evolutionary genetics provided me a strong background framework, on which multiple other acquired disciplines, forced by circumstances along my uneven journey, allowed me to grow intellectually, as well as making me aware of my limitations. As my late mentor, Dr. Van Rensselaer Potter said: “… humility with responsibility”. At the completion of my experimental journey, I continued to reflect on the implied meanings of my and others’ results to view the current state of collected knowledge on global health issues as one of all scientists’ obligations, namely, to weigh the value and bioethical implications of our work to global society. “

Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation James E. Trosko; From Radiation Genetics, Mutagenesis, Gap Junctions, Epigenetics, Stem Cells and an Integration of Radiation and Chemical Carcinogenesis. Radiat Res 2024; doi: https://doi.org/10.1667/RADE-24-00009.1 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search

Gap junctions (GJ) are suggested to support stem cell differentiation. The Muse cells that are applied in clinical trials are non-tumorigenic pluripotent-like endogenous stem cells, can be collected as stage-specific embryonic antigen 3 (SSEA-3+) positive cells from multiple tissues, and show triploblastic differentiation and self-renewability at a single cell level. They were reported to up-regulate pluripotency gene expression in suspension. We examined how GJ inhibition affected pluripotency gene expression in adherent cultured-Muse cells. Muse cells, mainly expressing gap junction alpha-1 protein (GJA1), reduced GJ intercellular communication from ~85% to 5–8% after 24 h incubation with 120 μM 18α-glycyrrhetinic acid, 400 nM 12-O-tetradecanoylphorbol-13-acetate, and 90 μM dichlorodiphenyltrichloroethane, as confirmed by a dye-transfer assay. Following inhibition, NANOG, OCT3/4, and SOX2 were up-regulated 2–4.5 times more; other pluripotency-related genes, such as KLF4, CBX7, and SPRY2 were elevated; lineage-specific differentiation-related genes were down-regulated in quantitative-PCR and RNA-sequencing. Connexin43-siRNA introduction also confirmed the up-regulation of NANOG, OCT3/4, and SOX2. YAP, a co-transcriptional factor in the Hippo signaling pathway that regulates pluripotency gene expression, co-localized with GJA1 (also known as Cx43) in the cell membrane and was translocated to the nucleus after GJ inhibition. Adherent culture is usually more suitable for the stable expansion of cells than is a suspension culture. GJ inhibition is suggested to be a simple method to up-regulate pluripotency in an adherent culture that involves a Cx43-YAP axis in pluripotent stem cells, such as Muse cells.

In this Commentary, the operational definition of cancer stem cells or cancer initiating cells includes the ability of certain cells, found in a heterogeneous mixture of cells within a tumor, which are able to sustain growth of that tumor. However, that concept of cancer stem cells does not resolve the age-old controversy of two opposing hypotheses of the origin of the cancer, namely the stem cell hypothesis versus the de-differentiation or re-programming hypothesis. Moreover, this cancer stem concept has to take into account classic experimental observations, techniques, and concepts, such as the multi-stage, multi-mechanism process of carcinogenesis; roles of mutagenic, cytotoxic and epigenetic mechanisms; the important differences between errors of DNA repair and errors of DNA replication in forming mutations; biomarkers of known characteristics of normal adult organ-specific stem cells and of cancer stem cells; and the characteristics of epigenetic mechanisms involved in the carcinogenic process. In addition, vague and misleading terms, such as carcinogens, immortal and normal cells have to be clarified in the context of current scientific facts. The ultimate integration of all of these historic factors to provide a current understanding of the origin and characteristics of a cancer stem cell, which is required for a rational strategy for prevention and therapy for cancer, does not follow a linear path. Lastly, it will be speculated that there exists evidence of two distinct types of cancer stem cells, one that has its origin in an organ-specific adult stem cell that is 'initiated' in the stem cell stage, expressing the Oct4A gene and not expressing any connexin gene or having functional gap junctional intercellular communication (GJIC). The other cancer stem cell is derived from a stem cell that is initiated early after the Oct4A gene is suppressed and the connexin gene is expressed, which starts early differentiation, but it is blocked from terminal differentiation.

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

The 'cancer stem cell' hypothesis has pointed to a specific target for new cancer therapies. The hypothesis is based on the observation that only the 'cancer stem cell' among the other heterogeneous cancer cells can sustain the growth of the cancer. The goal is to identify biomarkers of 'cancer stem cells' to distinguish them from the 'cancer non-stem cells' and normal adult tissue-specific stem cells. This analyst posits a hypothesis that, although all cancers originated from a single cell, there exist two types of 'cancer stem cells' either by the 'Stem Cell hypothesis' or from the 'De-differentiation hypothesis'. It is proposed that there exist two different 'cancer stem cells'. Some 'cancer stem cells' (a) lack the expression of connexins or gap junction genes and lack any form of gap junctional intercellular communication (GJIC) or (b) they have the expressed connexin-coded proteins for functional GJIC but are dysfunctional by some expressed oncogene. This is consistent with the Loewenstein hypothesis that a universal characteristic of cancer cells is they do not have growth control, nor terminally differentiate. This review speculates the normal organ-specific adult stem cell, that is 'initiated', is the origin of the 'cancer stem cells' with expressed Oct4A gene and no expressed connexin genes; whereas the other cancer stem cell has no expressed Oct4A genes but expressed connexin gene, whose coded protein is dysfunctional. Hence. both types of 'cancer stem cells' lack GJIC, for two different reasons, the selective therapies have to be different for these different cell types.

The International Agency for Research on Cancer (IARC) has recently proposed employing “ten key characteristics of human carcinogens” (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

Throughout the history of biological/medicine sciences, there has been opposing strategies to find solutions to complex human disease problems. Both empirical and deductive approaches have led to major insights and concepts that have led to practical preventive and therapeutic benefits for the human population. The classic definitions of “science” (to know) has been paired with the classic definition of technology (to do). One knew more as the technology developed, and that development was often based on science. In other words, one could do more if science could improve the technology. In turn, this made possible to know more science with improved technology. However, with the development of new technologies of today in biology and medicine, major advances have been made, such as the information from the Human Genome Project, genetic engineering techniques and the use of bioinformatic uses of sophisticated computer analyses. This has led to the renewed idea that Precision Medicine, while raising some serious ethical concerns, also raises the expectation of improved potential of risk predictions for prevention and treatment of various genetically and environmentally influenced human diseases. This new field Artificial Intelligence, as a major handmaiden to Precision Medicine, is significantly altering the fundamental means of biological discovery. However, can today’s fundamental premise of “Artificial Intelligence”, based on identifying DNA, as the primary nexus of human health and disease, provide the practical solutions to complex human diseases that involve the interaction of those genes with the broad spectrum of “environmental factors”? Will it be “precise” enough to provide practical solutions for prevention and treatments of diseases? In this “Commentary”, with the example of human carcinogenesis, it will be challenged that, without the integration of mechanistic and hypothesis-driven approaches with the “unbiased” empirical analyses of large numbers of data, the Artificial Intelligence approach with fall short.

Structures from the Stone Age can provide unique insights into Late Glacial and Mesolithic cultures around the Baltic Sea. Such structures, however, usually did not survive within the densely populated Central European subcontinent. Here, we ...The Baltic Sea basins, some of which only submerged in the mid-Holocene, preserve Stone Age structures that did not survive on land. Yet, the discovery of these features is challenging and requires cross-disciplinary approaches between archeology and ...

3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevantin vitromodels than traditional monolayer cultures, because they better mimicin vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro 'Spheroid_Finder' for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalianin vitrocell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

Growing evidence suggests dietary antioxidants reduce the risk of several cancers. Grape seeds extracts (GSE) are a rich source of polyphenols known to have antioxidant, chemopreventive and anticancer properties. Herein, we investigated the in vitro effects and putative action mechanisms of a grape seed extract (GSE) on human breast cancer cells (MCF-7). The effects of GSE were evaluated on cell proliferation, apoptosis and gap-junction-mediated cell-cell communications (GJIC), as basal mechanism involved in the promotion stage of carcinogenesis. GSE (0.05-100 μg/mL) caused a significant dose- and time-dependent inhibition of MCF-7 viability and induced apoptotic cell death, as detected by Annexin-V/Propidium Iodide. Concurrently, GSE induced transient but significant enhancement of GJIC in non-communicating MCF-7 cells, as demonstrated by the scrape-loading/dye-transfer (SL/DT) assay and an early and dose-dependent re-localization of the connexin-43 (Cx43) proteins on plasma membranes, as assayed by immunocytochemistry. Finally, real-time-PCR has evidenced a significant increase in cx43 mRNA expression. The results support the hypothesis that the proliferation inhibition and pro-apoptotic effect of GSE against this breast cancer cell model are mediated by the GJIC improvement via re-localization of Cx43 proteins and up-regulation of cx43 gene, and provide further insight into the action mechanisms underlying the health-promoting action of dietary components.

To protect the public's health from exposure to physical, chemical, and microbiological agents, it is important that any policy be based on rigorous scientifically based research. The concept of "linear no-threshold" (LNT) has been implemented to provide guideline exposures to these agents. The practical limitation to testing this hypothesis is to provide sufficient samples for experimental or epidemiological studies. While there is no universally accepted understanding of most human diseases, there seems to be better understanding of cancer that might help resolve the "LNT" model. The public's concern, after being exposed to radiation, is the potential of producing cancer. The most rigorous hypothesis of human carcinogenesis is the "multistage, multimechanism" chemical carcinogenesis model. The radiation carcinogenesis LNT model, rarely, if ever, built it into their support. It will be argued that this multistage, multimechanism model of carcinogenesis, involving the "initiation" of a single cell by a mutagen event, followed by chronic exposure to threshold levels of epigenetic agents or conditions that stimulate the clonal expansion of the "initiated" cell, can convert these benign cells to become invasive and metastatic. This "promotion" process can be interrupted, thereby preventing these initiated cells from transitioning to the "progression" process of invasion and metastasis.

Early observations showed a lack of growth control and terminal differentiation with a lack of gap junctional intercellular communication (GJIC). Subsequent observations showed that epigenetic tumor promoters and activated oncogenes, which block gap junction function, provide insights into the multi-stage, multi-mechanism carcinogenic process. With the isolation of embryonic induced pluri-potent stem cells and organ-specific adult stem cells, gap junctions were linked to early development. While tumors and tumor cell lines are a heterogeneous mixture of "cancer stem cells" and "cancer non-stem cells", the cancer stem cells seem to be of two types, namely, they express (a) no connexin genes or (b) connexin genes, but do not have functional GJIC. These observations suggest that these "cancer stem cells" originate from normal adult stem cells or from the de-differentiation or re-programming of somatic differentiated cells. This "Concept Paper" provides a hypothesis that "cancer stem cells" either originate from (a) organ-specific adult stem cells before the expression of the connexin genes or (b) organ-specific adult stem cells that just express gap junction genes but that the connexin proteins are rendered dysfunctional by activated oncogenes. Therefore, cancer prevention and therapeutic strategies must account for these two different types of "cancer stem cell".

The two hypotheses of the origin of cancer are the “stem cell” hypothesis and the “Dedifferentiation” or “Re-programming” hypothesis. Cancers are derived from a single cell, with all the cells within the tumor exhibiting heterogeneity of genotypes and phenotypes. Cancer cells, which do not contact inhibit; have growth control; terminally differentiate; are“immortal”; also, do not have function gap junctional intercellular communication (GJIC).The interpretation of animal experiments has suggested that the carcinogenic process consists of three phases, namely, the “initiation” phase; the “promotion” phase and the “progression” phase. With the isolation of embryonic-, induced pluripotent-, somatic nuclear transferand adult stem cells, observations have shown that stem cells express the embryonic gene, Oct4A, but not the gap junction genes. With the isolation of “side population” cells from cancer cell lines, which were shown to sustain the growth of tumors, the terms, “cancer-initiating” and “cancer stem cells”, were born. Use of human adult stem cells, expressing Oct4A, were shown to give rise to “initiated” Oct4Apositive cells which, after further modification, could give rise to “cancer stem cells”, whereas their differentiated daughters, not expressing Oct4A, never gave rise to “initiated” cells. There are “cancer stem cells” that do not express Oct4A. However, they do express their connexin gene but are unable to have functional GJIC. Therefore, the strategy to target “cancer stem cells” for cancer therapy must take recognize that there are two different “cancer stem cells” and therefore, two different approaches will have to be developed.

Cyanotoxins microcystin-LR (MC-LR) and cylindrospermopsin (CYN) represent hazardous waterborne contaminants and potent human hepatotoxins. However, in vitro monolayer cultures of hepatic cell lines were found to recapitulate, poorly, major hepatocyte-specific functions and inadequately predict hepatotoxic effects of MC-LR and CYN. We utilized 3-dimensional (3D), scaffold-free spheroid cultures of human telomerase-immortalized adult liver stem cells HL1-hT1 to evaluate hepatotoxic potential of MC-LR and CYN. In monolayer cultures of HL1-hT1 cells, MC-LR did not induce cytotoxic effects (EC50 > 10 micromol/L), while CYN inhibited cell growth and viability (48h-96h EC50 ≈ 5.5-0.6 micromol/L). Growth and viability of small growing spheroids were inhibited by both cyanotoxins (≥0.1 micromol/L) and were associated with blebbing and disintegration at the spheroid surface. Hepatospheroid damage and viability reduction were observed also in large mature spheroids, with viability 96h-EC50 values being 0.04 micromol/L for MC-LR and 0.1 micromol/L for CYN, and No Observed Effect Concentrations <0.01 micromol/L. Spheroid cultures of adult human liver stem cells HL1-hT1 exhibit sensitivity comparable to cultures of primary hepatocytes and provide a simple, practical, and cost-effective tool, which can be effectively used in environmental and toxicological research, including assessment of hepatotoxic potential and effect-based monitoring of various samples contaminated with toxic cyanobacteria.