Cell Proliferation Marker Research Articles

Hsa_circTCF25 Facilitates Oncogenesis in Gastric Cancer Cells by Modulating miR-149 Expression.

Circular RNA (circRNA) is related to gastric carcinogenesis and progression. This study explored the effects of circTCF25 on gastric cancer cell proliferation, migration, invasion, and cancer stem cell markers, as well as the potential network of circTCF25-miR and miR-149. circTCF25 expression was detected in tissue specimens and cells by real-time quantitative reverse transcription polymerase chain reaction. Cell Counting Kit-8 and transwell assays were used to measure the effects of circTCF25 knockdown on proliferation, migration and invasion. The potential network of circTCF25 was analyzed using bioinformatic analysis. circTCF25 was overexpressed in human gastric cancer tissues, and a series of cancer cell lines, and was associated with shorter overall survival. Interfering with circTCF25 reduced gastric cancer cell proliferation, migration, invasion and expression of cancer stem cell markers. CircTCF25 reduced expression of miR-149, apparently by acting as a miR-149 sponge. A new circTCF25-miR-149 competitive endogenous RNA network in gastric cancer was constructed, and most core genes were associated with the malignant growth and metastatic behavior of gastric cancer. circTCF25 may have prognostic value and an oncogenic role in gastric cancer. A circTCF25-miR-149 RNA regulatory network was established which may provide novel biomarkers or potential therapeutic targets for treating gastric cancer.

Background levels and brain organoid impact of RF field exposure in a healthcare environment

Introduction: This study is an introduction to the empirical and impact evaluation of radiofrequency electromagnetic field (RF-EMF) radiation exposure in a healthcare environment, focusing on an indoor microenvironment. It explores the expression of various genes associated with cellular responses, cell proliferation, senescence, and apoptotic cell death. The assessment analyzes current personal mobile communications (2G-5G FR1), providing a clear understanding of RF-EMF exposure and compliance with regulatory limits.Methods: The signals from different wireless communication systems at Hospital Universitario de Canarias (HUC) in Tenerife, Canary Islands, Spain, were examined in 11 locations. Four measurement campaigns were performed with frequency-selective exposimeters (PEMs) and an EME Spy 200 MVG, and experimental electric field values were compared as a long-term exposition. The frequency with the highest contribution (2.174 V/m) observed (1840 MHz) in UMTS was selected for biological effects evaluation.Results: The study focuses on four locations with the highest exposure to communication systems (downlinks), analyzing the results to verify compliance with regulations that ensure the safety of patients, the general public, and healthcare workers. LTE B20 (DL), GSM+UMTS 900 (DL), GSM 1800 (DL), UMTS 2100 (DL), and LTE B7 (DL) exhibited relatively higher E/m values throughout the campaigns, and these values consistently remained below the ICNIRP reference levels, signifying a consistently low level of exposure. In addition, this work presents the biological effects on neural stem cells (NSCs) using 3D brain organoids (BOs) exposed to RF signals in a validated and commercial experimental setting: the Gigahertz Transverse Electromagnetic cell (GTEM). The GTEM allows for the creation of homogeneous field electromagnetic fields in a small, enclosed setting and guarantees exposure conditions in a wide range of frequencies. BOs are an in vitro 3D cell-culture technology that reproduces the cellular composition and structure of the developing brain. Analyzing the expression of several genes associated with cellular responses, cell proliferation, senescence, and apoptotic cell death, we found that exposure of BOs at 1840 MHz did not affect mRNA expression in brain genes related to apoptosis or senescence. However, a decrease in gene expression for cell proliferation and cell activity markers was observed during the differentiation stage of BOs.Discussion: The discussion emphasizes the coexistence and evolution of various heterogeneous networks and services throughout the four measurement campaigns. Across all measured results, the levels of the obtained E-field were consistently well below the exposure limits set by internationally accepted standards and guidelines. These obtained values have been established in order to consider their potential effects on cell proliferation and cell activity, especially in differentiating biological organisms. Consequently, the results obtained and the methodology presented could serve as a foundational framework for establishing the basis of RF-EMF assessment in future heterogeneous 5G developments, particularly in the millimeter wave (mmWave) frequency range, where the forecast is for massive high-node density networks.

Results of Immunohistochemical Studies of Expression of Markers of Cell Cycle Regulation, Proliferation and Apoptosis in Different Types of Neoplasia of the Accessory Lub of the Mammary Gland

Results of Immunohistochemical Studies of Expression of Markers of Cell Cycle Regulation, Proliferation and Apoptosis in Different Types of Neoplasia of the Accessory Lub of the Mammary Gland

Abstract 3575: The estrogenic endocrine disrupting compounds bisphenol-a (BPA) and alpha-zeranol (aZAL) induce early mammary hyperplasia in female ACI rats

Abstract Endocrine disrupting compounds (EDCs) found in food-grade plastics, drinking water, and foodstuffs, pose a threat to human health. Estrogenic EDCs are capable of binding and activating the estrogen receptor (ER). Since breast tissue relies on estrogen signaling for growth and renewal, excessive estrogen stimulation is associated with breast cancer development. Breast cancer is a leading cause of cancer-related death in women. Only 5-10% of all breast cancer cases can be attributed to genetic causes, and the proportion of breast cancers of the luminal subtype are increasing in the United States for reasons unknown. Estrogenic EDCs may be able to initiate breast cancers through their estrogenic activities. The present study assessed two EDCs: the plasticizer bisphenol-A (BPA) and the mycotoxin alpha-zeranol (aZAL). To assess the estrogenic effects of these EDCs in an in vivo model, we utilized the ACI rat strain due to its sensitivity to estrogen-induced mammary carcinogenesis. The hypothesis of this work was that BPA and aZAL will induce mammary gland proliferation and estrogen signaling at early timepoints post-exposure. Estrogen, BPA, or aZAL were implanted into the backs of female ACI rats in silastic tubes at either a 9 gram doses. At a timepoint of 5 days post implantation, we saw no overt toxicity of our treatment. Both BPA and aZAL resulted in increased glandular proliferation and hyperplasia in a comparable manner to estrogen. Immunohistochemistry and qPCR revealed that the estrogen signaling marker progesterone receptor (PGR) was significantly upregulated in the estrogen, BPA, and aZAL treatment groups compared to control. The percent positivity of PGR staining in the mammary glands were 21.5% +/- 4.2%, 25.7% +/- 1.7%, 27.9% +/- 3.8%, and 8.18% +/- 2.5% respectively. Additionally, PCNA, a marker of cell proliferation, was significantly upregulated in the estrogen and aZAL treatment groups. The percent positivity of PCNA staining in the mammary glands were 7.67% +/- 2.56%, 49.6% +/- 5.2%, and 41.0% +/- 11.5% respectively. Additionally, the weight of the pituitary gland, an estrogen-sensitive endocrine organ, significantly increased in both the estrogen and aZAL treatment groups. Overall, this study demonstrated that the estrogenic EDCs BPA and aZAL induced mammary gland proliferation and hyperplasia in a comparable manner to estrogen. This is concerning due to the routine detection of these two compounds in human biosamples. More information is needed to assess the risk of exposure to these compounds as it pertains to breast cancer and other hormonal cancers. Citation Format: Cassandra Winz, Ba Liu, Caroline Xie, Shlok Rohatagi, Eric Li, Philip Furmanski, Nanjoo Suh. The estrogenic endocrine disrupting compounds bisphenol-a (BPA) and alpha-zeranol (aZAL) induce early mammary hyperplasia in female ACI rats [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 3575.

Abstract 6675: Dissecting the relationship between mucin 5AC and the lung/gut microbiome in the pathogenesis of K-ras mutant lung cancer

Abstract Non-small cell lung cancer, including its most common subtype, lung adenocarcinoma (LUAD), is one of the most prevalent and lethal forms of cancer worldwide. This is largely due to patients having progressed to an advanced stage of disease by the time of diagnosis. Thus, better understanding the pathogenesis of early LUAD is paramount for the identification of key targets and development of preventive and therapeutic strategies for clinical management of the disease. KRAS mutations are the most common oncogenic driver alteration in human LUAD. Previously, we have found that KRAS mutant LUAD patients whose tumors harbor high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, have significantly lower survival and poor prognosis. Mucins are membrane-bound and secreted hydrophilic proteins essential in clearance of allergens, pathogens, and debris in the lung airway. However, overproduction of mucins, such as MUC5AC, plays roles in the pathogenesis of airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Given the known effect of mucin polymers on bacterial growth and the immune microenvironment in the context of other airway inflammatory diseases, in this study, our objective is to better understand the MUC5AC-to-microbiome relationship using a lung epithelial specific mouse model of KRAS-mutant LUAD (CC-LR) and test the combinatorial effects of genetic deletion of MUC5AC (CC-LR/Muc5ac−/−) and the pan-microbiome depletion on tumor progression, the lung immune contexture, and microbiome (lung and gut). Thus far, we found that genetic deletion of MUC5AC or pan-microbiome depletion significantly decreases the tumor burden (66% vs 50%, respectively). Additionally, both groups show to have a significantly decreased percentage of tumor-specific Ki67+ cells, a cell proliferation marker, and ERG+ cells, an angiogenesis marker, in comparison to the CC-LR controls. The combination of the lack of MUC5AC with pan-microbiome depletion has also shown to reduce tumor development that needs further investigation. These findings highlight the need to better understand the mechanistic interaction between mucins and the lung/gut microbiome in the context of KRAS-mutant lung cancer which we are currently exploring. It also suggests that their relationship could be a key target for modulation, potentially leading to the development of a minimally invasive alternative preventive and/or treatment modality against KRAS-mutant LUAD. Citation Format: Maria T. Grimaldo, Michael J. Clowers, Avantika Krishna, Katherine E. Larsen, Nastaran Karimi, Yasmina H. Rezai, Arnav Gaitonde, Farbod Khalaj, Haoyue Liu, Florencia McAllister, Seyed Javad Moghaddam. Dissecting the relationship between mucin 5AC and the lung/gut microbiome in the pathogenesis of K-ras mutant lung cancer [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 6675.

Abstract 2073: Human breast patient derived organoids: Development and characterization of a new tissue mimetic model

Abstract Worldwide, breast cancer (BC), is the most frequently diagnosed cancer among women. In the US, 1 out of 8 women may expect to be diagnosed with BC In their lifetime. Current models for studying BC tumor biology and BC drug discovery include 2D cultured cell lines and patient derived xenografts (PDX). As 2D cultured cells do not represent the 3D complexity of human tissue, and PDX models are time consuming and cost prohibitive, better tissue mimetic models are needed. Organoids are self-assembled 3D cultured cellular units that may mimic the corresponding human tissue structurally and functionally (1). Patient derived organoids (PDO) are developed from tissue of specific patients and may resemble the source tissue in molecular features, including recapitulation of patient specific responses to therapies (2). Here, we report development and characterization of novel breast PDO lines from fresh (≤24h post-surgery) and cryopreserved (≤7 days post-surgery) tissue. Breast tumor and normal, tumor adjacent (NTA) tissue were sourced from consented patients. Breast PDO were generated following modifications to a published protocol (3). Briefly, tissue samples were minced, digested with collagenase, and filtered. The filtrate was resuspended in Matrigel (Corning) and small drops were added on cell culture plates. Both tumor and NTA PDO were cultured using the same media and organoids were monitored using brightfield microscopy (Olympus CK40) and live cell imaging (Muvicyte, Perkin Elmer). PDO lines were analyzed for expression and distribution of important breast-specific biomarkers using confocal microscopy (ImagExpress, Molecular Devices). PDO lines displayed hormone receptors (ER, PR), cell surface (EpCAM, CD49f), cytoskeletal (cytokeratin 14, 19, E-cadherin, vimentin) and cell proliferation (Ki67) markers. Transcriptome profiles were analyzed using vendor services (Azenta). PDO were also tested for their susceptibility to breast cancer drugs, e.g., Tamoxifen, chlorambucil and Docetaxel. We have successfully generated one NTA ER+/PR+/HER2− PDO line and one tumor ER+/PR+/HER2− PDO line from different donors. Our breast PDO lines would be highly useful in understanding tumor biology, BC progression, signal transduction, and drug discovery/testing. Future efforts would include process scaling up and development of BC specific assays.

Abstract 1821: Conessine inhibits cell proliferation, migration and invasion, and induces apoptosis in liver cancer cells

Abstract Cancer-related deaths from hepatocellular carcinoma (HCC) rank first worldwide. Although many attempts have been made to treat HCC, the survival rate remains unsatisfactory. Many plants from the family Apocynaceae, such as Holarrhena floribunda, contain conessine, a steroidal alkaloid. Several studies have indicated that conessine acts as an antihistamine with cancer therapy and antimicrobial resistance, but it hasn't been tested for its anti-cancer activity against liver cancer. Our study aimed to investigate the anticancer properties of conessine against liver cancer. We tested the effects of conessine on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) markers using both Huh7 and Mahlavu cells. Our experimental results indicated that conessine inhibited cell proliferation, migration, motility, and invasion in Huh7 and Mahlavu cells. Results of Western blot analysis showed that conessine induced apoptosis and EMT in liver cancer cells. Taken together, our results demonstrated that conessine exhibits anticancer activity in liver cancer through suppressing cell proliferation, migration, and invasion, and inducing apoptosis, suggesting it may have benefits for liver cancer treatment. Citation Format: Chia-Yang Li, Yu-Sheng Lin. Conessine inhibits cell proliferation, migration and invasion, and induces apoptosis in liver cancer cells [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 1821.

Abstract 1488: Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer associated with high rates of recurrence and metastasis, which contribute to poor overall survival. The challenges in treating TNBC stem from its high intratumor heterogeneity and lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 druggable targets. The tumor microenvironment (TME) plays a crucial role in disease progression. Within the TME, complex spatial interactions among tumor cells, immune cells, stroma, angiogenesis- and hypoxia-inducing factors collectively contribute to the advancement and spread of TNBC. In-depth understanding of the TME and interactions of the various components within can elucidate newer therapeutic targets, enhancing the prognosis of TNBC patients. Imaging mass cytometry (IMC) enables detailed analysis of cell-cell and cell-matrix interactions within the TME at the single-cell level. In this study, we aim to characterize the spatial relationships among various cell types within the TNBC TME using IMC, while also investigating differences in the TME of TNBC patients who developed distant metastasis (DM) compared to those who remained non-metastatic (NM). IMC was performed on formalin-fixed paraffin-embedded TNBC primary resection tissues (n= 16; DM= 7, NM= 9) from Stavanger University Hospital, Norway, using a panel of 25 metal-conjugated antibodies. Regions of interest were marked by the pathologist and the slides were laser ablated using Hyperion Imaging System. DeepCell was used to segment single cells based on surface markers. Mean intensity values of all markers for each cell were extracted and consolidated in R scripts for unsupervised clustering. Cell clusters were annotated based on phenotypic markers. Normalized expression of markers was compared for each cell type across DM and NM patients, including markers for cell proliferation, angiogenesis, hypoxia, and cell migration. Neighborhood analysis examined spatial associations between different cell types, highlighting differences in DM and NM patients. The IMC panel successfully provided spatial insights into the TME, including the characterization of tumor cells, immune cells (B cells, T cells, macrophages), endothelial cells, and proliferating cells. We identified 18 unique phenotypic cell clusters that were present at different frequencies in DM and NM patients. These clusters demonstrated different expression profiles of functional markers. t-SNE plots revealed distinct patterns of cell type distribution and unique protein expression profiles, highlighting significant differences between the two groups. Our data suggest a distinct spatial organization of the TME in TNBC patients who develop metastasis. This warrants further investigations to determine the role and interplay of TME features in predicting the risk of distant metastasis in TNBC. Citation Format: Mahak Bhargava, Jaspreet Kaur, Geetanjali Saini, Junjun Zheng, Yitian Xu, Shu-Hsia Chen, Ping-Ying Pan, Emma Rewcastle, Umay Kiraz, Emiel Adrianus Janssen, Ritu Aneja. Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer [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 1488.

Abstract 1631: A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture

Abstract We describe a novel homogeneous cell-based assay developed for the quantitative detection of Ki-67 protein, a recognized marker of cell proliferation, utilizing Lumit™ immunoassay technology. This luminescent assay has been effectively multiplexed with a fluorescent measure of cell death, employing a cell-impermeant fluorogenic DNA dye. This multiplex allows for the clear distinction between antiproliferative activity and overt cytotoxicity in cell culture models. Through varying lytic conditions, we demonstrated effective cell lysis and detection of nuclear Ki-67 protein. Specifically, Lumit™ Lysis Buffer II provided efficient recovery of Ki-67 protein without compromising assay performance, including the requisite binding of labeled antibodies to the target protein. This assay exhibited excellent signal-to-background (up to 40-fold) and signal linearity (R^2 = 0.99) in wildtype HeLa cells (~1000 to 80,000/well), while the signal in Ki-67 knockout HeLa cells was comparable to the no-cell background, indicating excellent specificity for Ki-67 protein. Due to the unavailability of recombinant full-length Ki-67 protein, two different recombinant Ki-67 protein fragments were used to corroborate assay sensitivity of <1 pM Ki-67 protein. The assay's suitability for human Ki-67 protein detection, not mouse, was observed through further cell-based testing. The proliferation/cytotoxicity assay multiplex was validated in both suspension and adherent cell lines, as well as primary cell culture, detecting antiproliferative and proliferative responses. CDK4/6 inhibitors are a class of antiproliferative agents of significant interest for their potential in treating cancer. In Jurkat cells, treatment with the CDK4/6 inhibitor palbociclib led to a dose-dependent reduction in Ki-67 protein expression with an EC50 of 88 nM, and total Ki-67 elimination at 2 μM compound concentration. Notably, there was no cytotoxicity detected at the tested doses and treatment duration (up to 48 h), but a significant reduction in viable cell number at maximal palbociclib concentrations, aligning with an antiproliferative mechanism. This work establishes a rapid, simple, luminescent assay for determining Ki-67 levels that can be paired with a fluorogenic readout of cell death, facilitating efficient and quantitative discernment of altered proliferation in mammalian cell culture using standard microplate readers. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture [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 1631.

Determination of the Ki-67 threshold value for predicting lymph nodes involvement in patients with gastric cancer

The search for biological markers to assess metastatic involvement of the lymph nodes in gastric cancer is one of the key steps in determining treatment tactics. The role of Ki-67 as a marker of cell proliferation in gastric cancer remains relevant. The aim of our study is to determine the Ki-67 threshold value for predicting the lymph nodes metastases. A retrospective analysis of 154 patients with gastric cancer showed an independent and statistically significant relationship between the depth of tumor invasion T (p = 0.002), the differentiation grade G (p = 0.010), the value of the Ki-67 index (p < 0.0001) and metastatic involvement of the lymph nodes. Using ROC analysis we found that Ki-67 ≥ 45 % correlates with the optimal level of sensitivity (55.9 %), specificity (84.2 %) and accuracy (73.4 %) of the method AUC 0.738 (p >< 0.043; 95 % CI 0,654–0,823). When evaluating the overall survival of patients>< 0.0001) and metastatic involvement of the lymph nodes. Using ROC analysis we found that Ki-67 ≥ 45 % correlates with the optimal level of sensitivity (55.9 %), specificity (84.2 %) and accuracy (73.4 %) of the method AUC 0.738 (p < < 0.043; 95 % CI 0,654–0,823). When evaluating the overall survival of patients >0.043; 95 % CI 0,654–0,823). When evaluating the overall survival of patients with Ki-67 > 45 %, we found that the median OS was 32 months [HR 2.2; 95 % CI 1.2–3,9; p = 0.005], while it was not reached in the group with Ki-67 < 45 %.A Ki-67 level of ≥ 45 % is the optimal threshold for determining the likelihood of lymph node metastasis in gastric cancer.

Hypoalbuminemia, but not derived neutrophil to lymphocyte ratio (dNLR), predicts overall survival in neuroendocrine tumours undergoing peptide receptor radionuclide therapy: A retrospective, cohort study of 557 patients.

Several inflammation scores have shown association with survival outcomes for patients with neuroendocrine tumours (NET) treated with peptide receptor radionuclide therapy (PRRT). However, whether these scores add value to established prognostic factors remains unknown. In this retrospective, cohort study of 557 NET patients undergoing PRRT in a tertiary referral centre from 2005 to 2015, we examined inflammatory markers and scores previously associated with cancer outcomes, using Cox proportional hazard models and Akaike's information criterion. Lower albumin (hazard ratio [95% confidence interval], .91 [.87-.95] per unit), as well as higher C-reactive protein (CRP; 1.02 [1.01-1.02]), Glasgow Prognostic Score (GPS; 1 vs. 0: 1.67 [1.14-2.44], 2 vs. 0 3.60 [2.24-5.79]), CRP/albumin ratio (1.84 [1.43-2.37]) and platelet count (Plt) × CRP, but not white blood cell, neutrophil and thrombocyte counts or derived neutrophil to lymphocyte ratio (dNLR), were associated with shorter median overall survival (OS) in an adjusted analysis. The addition of parameters based on albumin and CRP, but not dNLR, to a base model including age, chromogranin A, the cell proliferation marker Ki-67, performance status, tumour site and previous treatments improved the predictive accuracy of the base model. In an exploratory analysis of patients with available erythrocyte sedimentation rate (ESR) and CRP, ESR emerged as the most powerful predictor. When added to a prognostic model for OS in NET patients treated with PRRT, most inflammation scores further improved the model. Albumin was the single marker adding most value to the set of established prognostic markers, whereas dNLR did not seem to improve the model's prognostic ability.

Can the genetic background modulate the effects of feed additives? Answers from gut microbiome and transcriptome interactions in farmed gilthead sea bream (Sparus aurata) fed with a mix of phytogenics, organic acids or probiotics

The synergies between selective breeding and feed additives remain under-explored in farmed fish, despite their sustainability. Reference (REF) and selected gilthead sea bream for growth (GS) were fed with the control (CTRL) diet during 14 days. CTRL diet was oil-coated with three functional additives (PHY: phytogenic based on garlic and medium chain fatty acid; OA: organic acid mixture with a 70% of butyric acid sodium salt; PROB: probiotic based on Bacillus subtilis, pumillus and licheniformes species). These experimental diets were then sequentially administered at high (PHY/OA = 7.5 g/kg, PROB = 2 × 1011 CFU/kg; 2 weeks) and low (PHY = 5 g/kg, OA = 3 g/kg, PROB = 4 × 1010 CFU/kg; 10 weeks) additive doses. The capacity of a given genotype and additive to modify the fish growth performance, gut health and the host interaction with its anterior intestine (AI) microbiota was evaluated as a whole population or individually (9 fish/diet/genetics). GS fish showed a better growth and feed conversion ratio, linked to a reduced individual variability of gut microbial composition. The PHY additive had a major impact upon the intestinal transcriptome of GS-PHY fish, with the up-regulation of markers of epithelial integrity, sphingolipid and cholesterol/bile salt metabolism. With the OA additive, impaired growth performance, reduced AI goblet cell area and enhanced AI granulocyte infiltration were concomitant with a down-regulation of neutrophil degranulation markers associated with a decrease of pathogenic genera (Staphylococcus/Streptococcus/Neisseria), and an over-representation of acetone/butanol/ethanol fermentation and vitamin K biosynthesis inferred pathways. Bacillus establishment and lack of AI inflammation were parallel in PROB fish of both genetic backgrounds. However, GS fish grew and utilized feed better with the additive, whereas a worsening appeared in REF fish. This amelioration was related with a higher abundance of the nitrate-reducer Kocuria, an up-regulation of markers of epithelial cell maintenance and proliferation, and a down-regulation of microbiota-correlated protein synthesis and ubiquitination markers, supporting a reduced epithelial turnover and improved intestinal barrier function. Overall, the success of nutritional innovations in gilthead sea bream is largely dependent on the host genome predisposition, but also on the intestinal microbiota according to the hologenome theory.

Immunolocalization of telomerases in human hairs identifies proliferating cells in the bulb matrix and outer root sheath

Telomerase is present in cells with numerous or even un-limited replicative cycles, and some studies suggest it is a stemness marker. In order to determine whether this is the case for the human hair bulbs, an immunohistochemical and ultrastructural study has been carried out using antibodies against telomerase and PCNA (a cell proliferation marker). The observed labeling is similar for the two antibodies here utilized and is mainly nuclear. More frequent telomerase-positive cells are seen in the matrix epithelium of anagen hair bulbs but sparse labeled cells are also seen in the outer root sheath. In late catagen and also in telogen hair follicles only sparse labeled cells are present in the outer root sheath and few cells also in the secondary germinal epithelium formed at the base of the hair bulb in telogen. Electron microscopic immunogold shows a prevalent nuclear distribution and a lower cytoplasmic distribution in sparse cells of anagen bulb matrix that contain few keratin bundles. The nuclear localization is generally seen over the euchromatin or in areas occupied by more compact chromatin that may indicate an activity of telomerase in chromatin assemblage or dis-assemblage. The study concludes that the localization of telomerase is present in cells undergoing proliferation, namely transit amplifying cells of the outer root sheath that are sparsely detected in the lowermost secondary germinal hair bulb also in telogen.

Alterations in epidermal stem cells within the pilosebaceous unit in atrophic acne scars.

Atrophic acne scarring is a common sequela of inflammatory acne, causing significant problems for affected patients. Although prolonged inflammation and subsequent aberrant tissue regeneration are considered the underlying pathogenesis, the role of epidermal stem cells, which are crucial to the regeneration of pilosebaceous units, remains unknown. To examine the changes occurring in epidermal stem cells in atrophic acne scars. Changes in collagen, elastic fibre and human leukocyte antigen (HLA)-DR expression were analysed in normal skin and inflammatory acne lesions at days 1, 3 and 7 after development. The expression of epidermal stem cell markers and proliferation markers was compared between normal skin and mature atrophic acne scar tissue. In acne lesions, inflammation had invaded into pilosebaceous units over time. Their normal structure had been destructed and replaced with a reduced amount of collagen and elastic fibre. Expression of stem cell markers including CD34, p63, leucine-rich repeat-containing G protein-coupled receptor (LGR)6 and LGR5, which are expressed in the interfollicular epidermis, isthmus and bulge of hair follicles, significantly decreased in atrophic acne scar tissue compared to normal skin. Epidermal proliferation was significantly reduced in scar tissue. These findings suggest that as inflammatory acne lesions progress, inflammation gradually infiltrates the pilosebaceous unit and affects the resident stem cells. This disruption impedes the normal regeneration of the interfollicular epidermis and adnexal structures, resulting in atrophic acne scars.

Biogenic Zinc Oxide Nanoparticles Attenuate Acute Lymphoblastic Leukemia Cell Proliferation through Oxidative Stress and DNA Damage

Background: The most prevalent pediatric cancer is acute lymphoblastic leukemia (ALL). It is exceedingly challenging to treat recurrent diseases, and there aren’t many new medications available for children with disease resistance. The size-dependent anticancer effect of zinc oxide nanoparticles (ZnONPs) on T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT 3 is the central theme of this study. Methods: The leaf and bark extracts of Diospyros montana were subjected to nanoparticle (NPs) synthesis and characterized analytically to acquire ZnONPs. The ZnONPs were characterized using UV-Vis spectra, DLS, XRD, EDX, SEM, and TEM analysis. Then the ZnONPs were separated into two groups having <50 nm and 50-100 nm NPs sizes. In addition, MTT assay, dual staining, autophagy, DNA damage, and oxidative damage measurement were done to find out the anti-cancer effect of the ZnONPs on MOLT 3 cells. Results: The ZnONPs exhibited a size-dependent anticancer effect against MOLT 3 cells. The IC50 of ZnONPs of < 50 nm was found to be ~75 µg/mL while the IC50 of ZnONPs of 50-100 nm size was found to be ~102 µg/mL. Treatment with ZnONPs of <50 nm size decreased mitochondrial membrane potential, more than that with 50-100 nm ZnONPs. On the other hand, autophagy was found to be more prevalent in 50-100 nm treated ZnONPs when compared with < 50 nm size. However, treatment with both sizes of ZnONPs reduced cell proliferation markers such as ki67 positive cells while increasing 8-OHG and HDCF-DA positive cells. Conclusion: The obtained results portrayed that different-sized ZnONPs induce different modes of T-ALL cell death. Small-sized ZnONPs revealed higher efficacy, highlighting the size-dependent property. Further, this finding denotes that ZnONPs could be an effective anticancer agent against dreadful diseases like T-ALL, warranting further investigation.

Cucurbitacin B modulates M2 macrophage differentiation and attenuates osteosarcoma progression via PI3K/AKT pathway.

Osteosarcoma is a common malignant bone tumour characterised by an aggressive metastatic potential. The tumour microenvironment, particularly the M2-polarised macrophages, is crucial for tumour progression. Cucurbitacin B (CuB), a triterpenoid derivative, is recognised for its anti-inflammatory and antitumour properties. This study investigates CuB and its effect on M2 macrophage differentiation and osteosarcoma progression, aiming to contribute to new treatment strategies. In vitro, THP-1 monocytes were stimulated with PMA, IL-13 and IL-4 to induce differentiation into M2 macrophages. Additionally, the influence of CuB on the proliferation, migration and invasion of osteosarcoma cells in the context of M2 macrophages was scrutinised. Crucial signalling pathways, especially the PI3K/AKT pathway, affected by CuB were identified and validated. In vivo, the osteosarcoma model was employed to gauge the effects of CuB on tumour weight, lung metastasis, angiogenesis, cell proliferation and M2 macrophage markers. The results showed that CuB inhibited M2 macrophage differentiation, leading to reduced proliferation, migration and invasion of osteosarcoma cells. CuB manifested an inhibitory effect on the PI3K/AKT pathway during the differentiation of M2 macrophages. In mouse models, CuB markedly reduced the tumour weight and the number of lung metastases. It also reduced the expression of angiogenesis and cell proliferation markers in tumour tissues, decreased the quantity of M2 macrophages and their associated markers and pathway proteins. In conclusion, CuB impedes osteosarcoma progression by inhibiting M2 macrophage differentiation via the PI3K/AKT pathway, presenting the potential for therapeutic advancements in osteosarcoma treatment.

Evaluation of the Recombinant Bacterial Chitinases as Anti-proliferative and Anti-migratory Agents for the Human Breast Cancer Cell Line, MCF-7.

Chitinases, a glycosyl hydrolase family 18 members, have a wide distribution in both prokaryotes and eukaryotes, including humans. Regardless of the absence of endogenous chitin polymer, various chitinases and chitinase-like proteins (CLPs) have been reported in mammals. However, several other carbohydrate polymers, such as hyaluronic acid and heparan sulfate, show structural similarities with chitin, which could be a potential target of chitinase and CLPs. Heparan sulfate is part of the integral membrane proteins and involves in cell adherence and migration. Hence, to demonstrate the effect of chitinase on cancer cell progression, we selected two chitinases from Serratia marcescens, ChiB and ChiC, which function as exo- and endo-chitinase, respectively. The ChiB and ChiC proteins were produced recombinantly by cloning chiB and chiC genes from Serratia marcescens. The cell viability of the Michigan Cancer Foundation-7 (MCF-7) cells was studied using different concentrations of the purified recombinant proteins. Cell viability assay was performed using 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide and water-soluble tetrazolium salt, and the effect of ChiB and ChiC on cell proliferation was studied by clonogenic assay. The cell migration study was analysed by wound healing, transwell migration, and invasion assays. Cell cycle analysis of propidium iodide-stained cells and cell proliferation markers such as pERK1/2, pAKT, and SMP30 were also done. It was observed that both ChiB and ChiC were able to impede cell viability, cell migration, and invasion significantly. These observations and our in silico molecular docking analysis suggest that ChiC is a potential anticancer agent and is more efficient than ChiB. Since the ChiC is able to inhibit both cancer cell proliferation and migration, it could be a potential candidate for the treatment of metastatic cancer.

Evaluation of the in vitro human skin percutaneous absorption of ketoprofen in topical anhydrous and aqueous gels.

Ketoprofen is a nonsteroidal anti-inflammatory drug used for the treatment of acute and chronic pain associated with inflammatory conditions. This study aims to evaluate the in vitro percutaneous absorption of ketoprofen 10% formulated in proprietary anhydrous and aqueous gels using the Franz skin finite dose model. The anhydrous gel was initially characterized for cytotoxicity using EpiDerm skin tissue model by cell proliferation assay and Western blot analysis. The Ultra Performance Liquid Chromatography method for measuring ketoprofen was validated and the stability of ketoprofen 10% in the anhydrous gel formulation was evaluated at 5°C and 25°C for 181 days. The percutaneous absorption of ketoprofen was determined using donated human skin. The tissue sections were mounted within Franz diffusion cells. A variable finite dose of each ketoprofen formulation in either anhydrous or aqueous gel was applied to the skin sections and receptor solutions were collected at various time points. Cell proliferation assay showed minimal cell death when EpiDerm skin tissue was exposed to the anhydrous gel for 24h; the levels of protein markers of cell proliferation were not affected after 17-h exposure. Ketoprofen was stable in the anhydrous gel when stored at 5°C and 25°C. When compounded in the anhydrous and aqueous gels, ketoprofen had mean flux rate of 2.22 and 2.50μg/cm2 /h, respectively, after 48h. The drug was distributed to the epidermis and dermis sections of the skin. Both the anhydrous and aqueous gels facilitated the percutaneous absorption of ketoprofen without statistically significant differences. The anhydrous gel can be used as a base to facilitate the transdermal delivery of ketoprofen. Although the anhydrous and aqueous gels can deliver a similar amount of ketoprofen, the anhydrous gel (water activity below 0.6) allows for extended default beyond-use-date of compounding preparations.

Prognostic value of the expression and localization of cell proliferation and apoptosis markers in unicystic ameloblastomas

The aim of this study was to verify whether the expression of cell proliferation and apoptosis markers in different types of unicystic ameloblastoma (UA) is associated with the location of neoplastic cells. Immunohistochemical study with a sample of 32 cases of UA, 11 cases of conventional ameloblastoma (CAM) and ten dental follicles (DF) cases was performed. Cell proliferation was assessed using Ki-67 status, and apoptosis by caspase-3 expression. Mural UA (MUA) showed a higher immunostaining of Ki-67 (p < 0.05) and a lower immunostaining of Caspase-3 (p < 0.05) compared with luminal and intraluminal subtypes of UA and CAM. The neoplastic cells of the MUA’s cystic capsule showed a higher expression of Ki-67 protein (p < 0.0001) and a lower expression of Caspase-3 (p < 0.0001) compared with the lumen. DF showed lower Ki-67 and Caspase-3 immunostaining (p < 0.05) than neoplasms. The higher immunoexpression of Ki-67 and the lower immunoexpression of Caspase-3 in MUA, in the parenchyma cells within the cystic capsule, suggest an association between the biological behaviour and location of neoplastic cells in a tumour.

PX-478, an HIF-1α inhibitor, impairs mesoCAR T cell antitumor function in cervical cancer.

Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated remarkable success in treating hematological malignancies. However, its efficacy against solid tumors, including cervical cancer, remains a challenge. Hypoxia, a common feature of the tumor microenvironment, profoundly impacts CAR T cell function, emphasizing the need to explore strategies targeting hypoxia-inducible factor-1α (HIF-1α). In this study, we evaluated the effects of the HIF-1α inhibitor PX-478 on mesoCAR T cell function through in-silico and in vitro experiments. We conducted comprehensive analyses of HIF-1α expression in cervical cancer patients and examined the impact of PX-478 on T cell proliferation, cytokine production, cytotoxicity, and exhaustion markers. Our in-silico analyses revealed high expression of HIF-1α in cervical cancer patients, correlating with poor prognosis. PX-478 effectively reduced HIF-1α levels in T and HeLa cells. While PX-478 exhibited dose-dependent inhibition of antigen-nonspecific T and mesoCAR T cell proliferation, it had minimal impact on antigen-specific mesoCAR T cell proliferation. Notably, PX-478 significantly impaired the cytotoxic function of mesoCAR T cells and induced terminally exhausted T cells. Our results underscore the significant potential and physiological relevance of the HIF-1α pathway in determining the fate and function of both T and CAR T cells. However, we recognize the imperative for further molecular investigations aimed at unraveling the intricate downstream targets associated with HIF-1α and its influence on antitumor immunity, particularly within the context of hypoxic tumors. These insights serve as a foundation for the careful development of combination therapies tailored to counter immunosuppressive pathways within hypoxic environments and fine-tune CAR T cell performance in the intricate tumor microenvironment.