Proliferation Marker Ki-67 Research Articles

Identification of the proliferative activity of germline progenitor cells in the adult ovary of the bat Artibeus jamaicensis.

Until a few years ago, it was assumed that oocyte renewal did not take place in the ovary of adult organisms; however, the existence of germline progenitor cells (GPCs), which renew the ovarian follicular reserve, has now been documented in mammals. Specifically, in the adult ovary of bats, the presence of cells located in the cortical region with characteristics similar to GPCs, called adult cortical germ cells (ACGC), has been observed. One of the requirements that a GPC must fulfil is to be able to proliferate mitotically, so the evaluation of cell proliferation in ACGC is of utmost importance in order to be able to relate them to a parental lineage. Currently, there are several methods to determine cell proliferation, including BrdU labelling or the use of endogenous proliferation markers. Thus, the aim of this work was to evaluate the proliferative activity of ACGC in the adult ovary of the bat Artibeus jamaicensis, using different proliferation markers and correlating these with the protein expression of the transcription factor Oct4 and the germ line marker Ddx4. We found that the expression pattern of the proliferation markers BrdU, PCNA, Ki-67 and pH3 occurs at different times of the cell cycle, so co-localization of two or more of these markers allows us to identify proliferating cells. This allowed us to identify ACGC with proliferative capacity in the adult ovary of A. jamaicensis, suggesting that GPCs renew the follicle reserve during the adult life of the organism.

Anticancer Activity of Vitamin D, Lumisterol and Selected Derivatives against Human Malignant Melanoma Cell Lines.

Despite the recent development of improved methods of treating melanoma such as targeted therapy, immunotherapy or combined treatment, the number of new cases worldwide is increasing. It is well known that active metabolites of vitamin D3 and lumisterol (L3) exert photoprotective and antiproliferative effects on the skin, while UV radiation is a major environmental risk factor for melanoma. Thus, many natural metabolites and synthetic analogs of steroidal and secosteroidal molecules have been tested on various cancer cells and in animal models. In this study, we tested the anti-melanoma properties of several natural derivatives of vitamin D3 and L3 in comparison to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). A significant decrease in melanoma cell proliferation and cell mobility was observed for selected derivatives, with (25R)-27-hydroxyL3 showing the highest potency (lowest IC50) in A375 cells but lower potency in SK-MEL-28 cells, whereas the parent L3 failed to inhibit proliferation. The efficacy (% inhibition) by 1,24,25(OH)3D3 and 1,25(OH)2D3 were similar in both cell types. 1,25(OH)2D3 showed higher potency than 1,24,25(OH)3D3 in SK-MEL-28 cells, but lower potency in A375 cells for the inhibition of proliferation. As for 1,25(OH)2D3, but not the other derivatives tested, treatment of melanoma cells with 1,24,25(OH)3D3 markedly increased the expression of CYP24A1, enhanced translocation of the vitamin D receptor (VDR) from the cytoplasm to the nucleus and also decreased the expression of the proliferation marker Ki67. The effects of the other compounds tested were weaker and occurred only under certain conditions. Our data indicate that 1,24,25(OH)3D3, which has undergone the first step in 1,25(OH)2D3 inactivation by being hydroxylated at C24, still shows anti-melanoma properties, displaying higher potency than 1,25(OH)2D3 in SK-MEL-28 cells. Furthermore, hydroxylation increases the potency of some of the lumisterol hydroxy-derivatives, as in contrast to L3, (25R)-27(OH)L3 effectively inhibits proliferation and migration of the human malignant melanoma cell line A375.

13-cis Retinoic Acid-Mediated Modulation of Human Meibomian Gland Epithelial Cells Development: Implications for In Vitro Modeling of Meibomian Gland Dysfunction.

Purpose: This study aimed to investigate the effect of 13-cis retinoic acid (13-cis RA) on human meibomian gland epithelial cells (HMGECs) and explore the potential of using this experimental model as an in vitro approach for studying meibomian gland dysfunction (MGD). Methods: First, HMGECs were cultured with 13-cis RA at different doses and times, and cell viability and proliferation rates were assessed to determine the appropriate stimulation concentration and time. Subsequently, during the proliferation stage, the expression of proliferation, inflammation, and oxidative stress genes and their products were evaluated. The meibum synthesis capacity was determined during the differentiation stage. Additionally, the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 was used as a control to assess the impact of 13-cis RA on PPARγ. Results: 13-cis RA significantly inhibited cell viability and proliferation in a time-dose response manner. Under the stimulation of 2 and 5 μM for 48 h during the proliferation stage, a significant decrease was observed in the expression of cell proliferation markers Ki67, antioxidant SOD-2, and Nrf-2. However, the expression of the pro-inflammatory factors IL-1β, IL-8, MMP9, and oxidative stress markers NOX-4 and reactive oxygen species increased. During the differentiation stage, it suppressed meibum synthesis and the expression of meibocyte differentiation-related proteins adipose differentiation-associated protein 4 (ADFP4), elongation of very long chain fatty acid protein 4 (ELOVL4), sterol regulatory element-binding protein 2 (SREBP-2), and PPARγ. Conclusion: 13-cis RA inhibited cell viability, promoted inflammation and oxidative stress, and suppressed meibum synthesis through the PPARγ pathway. Our study shed light on the effect of 13-cis RA on HMGECs and provided a promising direction for studying MGD in vitro.

7092 Blocking of Aggressive Tumor Progression of Anaplastic Thyroid Cancer by p53

Abstract Disclosure: E. Hwang: None. R. Chari: None. T. Kimura: None. S. Cheng: None. Background: Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer, with very limited treatment options. Patients succumb to death within 6-12 months after diagnosis. It has been observed that mutations of p53, occurring as tumorigenesis progresses, is associated with the aggressiveness of ATC. The present studies tested the hypothesis that wild type p53 (WTp53) could block tumor progression and mitigate its aggressiveness. Methods: We used human 8505C cells as a model, derived from human ATC tumors, harboring BRAFV600E mutation and one allele of mutated p53C742G. We re-expressed WTp53 or mutant p53C742G into 8505C cells (8505C-WTp53 or 8505C-MTp53 cells, respectively) via lentiviral transduction. We analyzed the effects of exogenous expressed WTp53 and mutant p53 on cell proliferation, apoptosis, and migration in vitro and the progression of tumors in vivo mouse xenograft models. Results: We showed that p53 proteins were more abundantly expressed in 8505C-WTp53 or 8505C-MTp53 cells than the parental 8505C cells. Using WTp53 luciferase reporter (PG-13), we demonstrated that the expressed WTp53 was functional as the transcription activity of p53 was higher than the parental 8505C cells. Consistently, in 8505C-MTp53, the reporter activity of PG-13 was not detected. The expressed WTp53 inhibited cell proliferation and decreased cell migration. Immunohistochemical analysis (IHC) showed decreased proliferation marker Ki67 and increased cleaved caspase-3, indicating the induction of apoptosis in 8505C-WTp53 cells. Importantly, we found that the NIS expression was increased in 8505C-WTp53 cells, but not in 8505C-MTp53 cells, as compared with 8505C cells. Xenograft studies showed that tumor progression was inhibited in nude mice injected with 8505C-WTp53 cells as compared with tumors induced by 8505C parental cells and 8505C-MTp53 cells. Consistent with in vitro findings, IHC demonstrated decreased proliferation marker Ki67 and increased cleaved caspase-3 proteins in tumors induced by 8505C-WTp53 cells. Furthermore, the expression of apoptotic regulators such as BAX and PUMA, known as p53 target genes, were elevated in 8505C-WTp53 cultured cells and in 8505C-WTp53-induced tumors, indicating induction of apoptosis. Conclusions: Our findings showed that the aggressiveness of ATC, caused by mutant p53 in late stage of tumor progression, can be mitigated by exogenous expression of WTp53. These results provided new insights into the deleterious actions of mutation of p53 in ATC tumor development. Importantly, our studies revealed that targeting p53 in ATC could be considered as a potential therapeutic opportunity. Presentation: 6/3/2024

Weitiao No. 3 (3) enhances the efficacy of anti-programmed cell death protein-1 immunotherapy by modulating the intestinal microbiota in an orthotopic model of gastric cancer mice.

To explore the effects of Weitiao No. 3 (3, WD-3) on anti-programmed cell death protein-1 (PD-1) immunotherapy in gastric cancer (GC). The intestinal microbiota was analyzed by 16S rDNA sequencing of fecal samples from three groups: healthy people (Health), GC patients (GC), and WD-3-treated GC patients (WD-3). Next, we established an orthotopic model of GC mice, which were treated with anti-PD-1, WD-3, or an inoculation of intestinal bacteria. Immune markers CD3, CD4, CD8, and forkhead box protein P3 (FOXP3), and the cell proliferation marker Ki67, were evaluated by immunohistochemistry. Cell apoptosis in GC tumors was assessed by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling staining. Enzyme-linked immunosorbent assays (ELISAs) were performed to analyze the serum levels of the following cytokines in GC mice: tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-6, IL-10, interferon (IFN)-γ, and transforming growth factor (TGF)-β. Sequencing data showed that there were significant differences in the composition of the gut microbial community among the three human groups. The gut bacteria in the three groups mainly comprised the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. At the genus level, the relative abundances of Bifidobacterium and Coprococcus showed significant decreases in the GC group, and an obvious increase in the WD-3 group, compared with the Health group. Interestingly, the relative abundance of Saccharopolyspora was only detected in the WD-3 group. The results of in vivo experiments in GC mice showed that WD-3 or anti-PD-1 treatment increased the levels of CD3+, CD4+, and CD8+ T cells, but decreased the levels of FOXP3+ regulatory T cells. Furthermore, WD-3 or PD-1 antibody treatment inhibited proliferation and promoted apoptosis of GC tumor cells. ELISA analysis showed that WD-3 or PD-1 antibody treatment facilitated TNF-α, IL-2, and IFN-γ expression, while suppressing IL-6, IL-10, and TGF-β expression. Combination therapy with WD-3 and anti-PD-1 intensified all of these effects. WD-3 enhanced the immunotherapeutic efficacy of anti-PD-1 by modulating the intestinal microbiota in an orthotopic model of GC mice.

5-aminosalicylic acid alleviates colitis and protects intestinal barrier function by modulating gut microbiota in mice.

5-aminosalicylic acid (5-ASA) is widely used in the treatment of ulcerative colitis (UC), but its anti-inflammatory mechanism is complex and has not been fully understood. DSS model was used to test the effect of 5-ASA. Tight junction and Ki-67 were detected by western blot, immunofluorescence, and immunohistochemistry or qPCR. 16S rRNA gene sequencing of gut microbiota and subsequent bioinformatics and statistical analysis were performed to identify the specific bacteria which were associated with the treatment effect of 5-ASA. GC-MS was performed to test short-chain fatty acids (SCFAs). Antibiotic-treated mice were used to demonstrate the key role of endogenous gut microbiota. Here, we found that 5-ASA alleviated dextran sulfate sodium (DSS)-induced colitis in mice. Moreover, 5-ASA significantly repaired the intestinal barrier. At the molecular level, 5-ASA markedly raised the expression of tight junction proteins including JAM-A and occludin and cell proliferation marker Ki-67 in mice. In addition, bacterial 16S rRNA gene sequencing and bioinformatics analysis showed that 5-ASA significantly modulated the DSS-induced gut bacterial dysbiosis. In detail, it stimulated the growth of protective bacteria belonging to Faecalibaculum and Dubosiella, which were negatively correlated with colitis parameters, and blocked the expansion of pro-inflammatory bacteria such as Escherichia-Shigella and Oscillibacter, which were positively correlated with colitis in mice. Meanwhile, 5-ASA increased the cecal acetate level. Most notably, 5-ASA was no longer able to treat colitis and reverse gut barrier dysfunction in antibiotic-treated mice that lacked endogenous gut microbiota. Our data suggested that the anti-inflammatory activity of 5-ASA required the inherent intestinal flora, and the gut microbiota was a potential and effective target for the treatment of ulcerative colitis.

Blockade of Src signaling prevented stemness gene expression and proliferation of patient-derived gastric cancer stem cells

ABSTRACT Objectives: Gastric cancer (GC) is one of the most malignant tumors. Mounting studies highlighted gastric cancer stem cells (GCSCs) were responsible for the failure of treatment due to recurrence and drug resistance of advanced GC. However, targeted therapy against GCSC for improving GC prognosis suffered from lack of suitable models and molecular targets in terms of personalized medicine. To address this issue, two patient-derived GC cell lines SD209 and SD292 with cancer stem cells (CSCs) such as phenotype were isolated for establishing targeted therapy aiming at critical metastatic signaling in GC. Materials and Methods: The primary patient-derived GCSCs were established from parts of GC tissues for characterization of stem cells (SCs) phenotype at both cellular and molecular levels. Western blot and Immunohistochemistry (IHC) were performed for identifying the deregulated signaling in GC tissue. Immunofluorescence was used for analyzing proliferating and SC markers in GCSC attached on fibroblast. Acridine orange and propidium iodide analyses were performed for the survival of GCSC in suspensions. Results: In the culture environments of both SD209 and SD292, a lot of mesenchymal fibroblasts spread and crowd together on which a lot of cell clumps, suspected as GCSC, were firmly attached. In the IHC analysis, the GCSC stemness genes CD44 and Ep-CAM increased in tumor tissues of SD209, whereas Nanog-1 and octamer-binding transcription factor 3 (OCT-3) increased in that of SD292. By immunofluorescent analysis of a proliferation marker Ki67, the growth of SD209 and SD292 on mesenchymal fibroblasts was found to be reduced by dasatinib, the inhibitor of the Src kinase whose activity was upregulated in tumor tissues of both GCs. Dasatinib also suppressed the expression of Nanog-1 and OCT-3 in SD292 attached on mesenchymal fibroblasts. Conclusion: This study may provide a base for targeted therapy against GCSCs/GCs progression in future preclinical/clinical settings.

Markers of epithelial-mesenchymal transformation in placentas from very early preterm births

Premature birth complicates one in ten births in the world, two-thirds of which are caused by spontaneous labor and premature rupture of membranes (PROM). The opinion about the inflammatory origin of preterm birth is generally accepted, more and more attention is devoted to the phenomena of epithelial-mesenchymal transformation (EMT).The objective: to study markers of cell proliferation and EMT in samples of placentas from very early preterm, early preterm and term births.Materials and methods. Placenta samples were examined from 101 women who had labour at 22–27 weeks of gestation on the background of PROM (I group), 102 women who had labour at 22–27 weeks on the background of intact fetal membranes (II group), 100 women who had labour at 28-34 weeks on the background of PROM (III group) and 102 women in labour – at 28-34 weeks on the background of intact membranes (IV group). Samples of 60 placentas from timely normal births were included in the control group.The presence of proliferation factor Ki-67 was determined in placenta samples by immunohistochemical method, cytokeratin-18 content was determined in 10 placentas of each group, and vimentin – in 10 amniotic membrane samples. Regarding the proliferation antigen Ki-67, the placentas were divided according to the detection of the sign in the distal, proximal and basal villi. The expression of cytokeratin and vimentin in syncytium and amnion cells was measured in conventional units and compared between groups.The statistical evaluation of the obtained differences was carried out using the Student’s test.Results. Proliferation antigen Ki-67 was detected in 61.9% of all placentas, including term births. In the groups of very early preterm birth (PB), they were detected mainly in villi of distal localization – in 44.6% in the group of PROM and in 51.0% – in the group of labour on the background of intact membranes. In the case of deliveries on the background of intact membranes in extremely premature terms, only 22.5% of the samples had a positive antigen in the basal villi, on the background of PROM in these terms – in 36.7%, in the groups of early PB – in 79.4% and 74.0% respectively.Distal Ki-67 expression in such placentas was a rare finding – 10.0% in the III group, 11.8% – in the IV group, and 8.3% – in placentas from term deliveries.In all gestational periods, the expression of cytokeratin in amniotic membranes in placentas from births on the background of PROM is statistically lower than in placentas from births on the background of intact membranes.In the case of very early PB, the greater expression of vimentin in the distal villi (0.324±0.001 units in the I group and 0.356±0.007 units in the II group) than in the intermediate villi (0.234±0.004 units and 0.248±0.002 units, respectively) and basal ones (0.178±0.002 units and 0.189±0.006 units, respectively). This once again confirms that the inflammatory reaction and the associated EMT at birth before 28 weeks are mainly of fetal origin.In placentas from early PB, the pattern is the opposite – from 0.345±0.007 units and 0.369±0.009 units in III and IV groups, respectively, in basal villi to 0.257±0.004 units and 0.239±0.005 – in intermediate villi and 0.178±0.009 units and 0.165±0.005 units – in the distal ones.Conclusions. 1. The expression of the inflammatory proliferation marker Ki-67 was detected in two thirds of the studied placentas regardless of the term of delivery, but very early PBs are characterized by the predominant localization of the marker in the distal villi, while in early PBs – in the basal and proximal villi. 2. The expression of EMT markers indicates the fetal origin of the inflammatory response in very early PB – less cytokeratin in syncytial cells and more vimentin in distal chorionic villi. In placentas from early PB and term deliveries, a more pronounced expression of vimentin in the basal vessels of the chorion dominates.

Mammary carcinoma in a male cat following long-term medroxyprogesterone acetate treatment: case report and review of the literature.

In male cats, as in men, mammary carcinomas are rarely reported. However, like in females, hormonal therapy is a significant risk factor. This study reports the case of an 11-year-old male cat with multiple mammary tumours and a history of long-term medroxyprogesterone acetate therapy for the suppression of sexual behaviour, along with a brief review of the literature. Complete surgical removal of the right mammary chain and the ipsilateral inguinal lymph nodes was performed, and all tissues were submitted for histology. Histological examination revealed the presence of a tumour in the third and fourth mammary glands, consisting of neoplastic cells arranged in various structures, including tubulopapillary and tubular structures, sometimes cystically dilated, and solid areas. The inguinal lymph nodes were also involved. The morphology was consistent with a diagnosis of mammary carcinoma, tubulopapillary type, with nodal metastases. Immunohistochemistry revealed that tumour cells were positive for cytokeratin (clones AE1/AE3), while stromal cells were positive for vimentin (clone V9). The proliferation marker Ki-67, evaluated on both the primary tumour and the nodal metastases, was strongly expressed in the nuclei of neoplastic cells, with a Ki-67 proliferation index of 8.9% and 20% for the primary tumour and the metastases, respectively. This case highlights the importance of considering the possibility of malignant mammary tumours not only in female but also in male cats with a history of long-term hormonal treatment for suppression of sexual behaviour.

Unveiling the antitumor synergy between pazopanib and metformin on lung cancer through suppressing p-Akt/ NF-κB/ STAT3/ PD-L1 signal pathway

Pazopanib, an inhibitor of the VEGF receptor tyrosine kinase, has demonstrated significant antitumor effects in lung cancer. However, its application as a standard treatment for this type of cancer is limited by its drug resistance and toxicity. Metformin has the potential to combat lung cancer by modifying the tumor’s immune microenvironment.In this study, we investigated the potential antitumor effects and the associated underlying molecular mechanisms of the combination of pazopanib and metformin in lung cancer. In vitro studies were conducted using the A549 and H460 lung cancer cell lines, whereas urethane-induced lung cancer-bearing mice were used for in vivo assessments. The urethane-induced mice received oral administration of pazopanib (50 mg/kg) and/or metformin (250 mg/kg) for a duration of 21 days.The results indicated that the MTT assay demonstrated a combined cytotoxic effect of the pazopanib/metformin combination in H460 and A549 cells, as evidenced by CI and DRI analyses. The observed increase in annexin V levels and the corresponding increase in Caspase-3 activity strongly suggest that this combination induced apoptosis.Furthermore, the pazopanib/metformin combination significantly inhibited the p-Akt/NF-κB/IL-6/STAT3, HIF1α/VEGF, and TLR2/TGF-β/PD-L1 pathways while also increasing CD8 expression in vivo. Immunohistochemical analysis revealed that these antitumor mechanisms were manifested by the suppression of the proliferation marker Ki67. In conclusion, these findings revealed that metformin augments the antitumor efficacy of pazopanib in lung cancer by simultaneously targeting proliferative, angiogenic, and immunogenic signaling pathways, metformin enhances the antitumor effectiveness of pazopanib in lung cancer, making it a promising therapeutic option for lung cancer.

Expression of Axl Receptor Tyrosine Kinase and Its Association With Ki-67 Proliferation Marker, BCL-2 Anti-apoptotic Protein, Hormone Receptor Status, and HER2/Neu Status in Breast Cancer Among Women From Duhok, Iraq.

Background Breast cancer (BC) is the most prevalent cancer among women worldwide, contributing to high mortality rates, especially in Iraqi women. Detecting the disease before metastasis may increase survival chances for many patients, but that is not the case for most of them. Thus the search for new prognostic biomarkers or testing the relevance of existing ones could contribute to therapeutic decisions complementing the traditional methods, including TNM (tumor, node, and metastasis) staging, tumor grade, and other clinicopathological features in addition to the use of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu). The Axl receptor is frequently associated with invasion, migration, poor prognosis, and angiogenesis. Furthermore, its association with chemotherapyand targeted therapy resistance makes itan ideal biomarker for therapeutic targeting. Methodology This study involved 50 malignant cases with 25 benign fibroadenoma and non-neoplastic cases represented by inflammatory conditions, collected with their corresponding data from the central lab in Duhok Governorate, Iraq. Expression of Kiel 67 (Ki-67) proliferation marker and B-cell lymphoma 2 (BCL-2) anti-apoptotic protein was measured using immunohistochemistry (IHC) to estimate tumor growth and apoptosis. Gene expression of the Axl receptor was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results Cases with high Ki-67 accounted for 68% and low Ki-67 cases were 32% across the graded groups and were significantly associated with tumor grade, PR, and HER2. BCL-2-negativecases accounted for 62% and BCL-2-positive cases were 38%. It was revealed that BCL-2 had a strong correlation with age, especially inthose under 50 years.As for the Axl gene expression, the average fold change in expression in the high-grade (H.) group was 1.74 times higher than in the control group, while in the low/intermediate (L.) group, it was 3.74 times higher. Additionally, when comparing these results with other variables, no significant associations were observed. Conclusion Axl receptor was not associated with all of the clinicopathological variables, the expression values were high in malignant tumors in comparison with the benign tumors, and it was found that Axl receptor expression was associated with low/intermediate grade, which is considered a favorable prognostic factor. Although Axl receptor expression was previously linked with proliferation and invasiveness in BC, its association with the Ki-67 proliferation marker and BCL-2 anti-apoptotic protein was not observed.

On-treatment biopsies to predict response to neoadjuvant chemotherapy for breast cancer

BackgroundPatients with pathologic complete response (pCR) to neoadjuvant chemotherapy for invasive breast cancer (BC) have better outcomes, potentially warranting less extensive surgical and systemic treatments. Early prediction of treatment response could aid in adapting therapies.MethodsOn-treatment biopsies from 297 patients with invasive BC in three randomized, prospective neoadjuvant trials were assessed (GeparQuattro, GeparQuinto, GeparSixto). BC quantity, tumor-infiltrating lymphocytes (TILs), and the proliferation marker Ki-67 were compared to pre-treatment samples. The study investigated the correlation between residual cancer, changes in Ki-67 and TILs, and their impact on pathologic complete response (pCR) and disease-free survival (DFS).ResultsAmong the 297 samples, 138 (46%) were hormone receptor-positive (HR+)/human epidermal growth factor 2-negative (HER2−), 87 (29%) were triple-negative (TNBC), and 72 (24%) were HER2+. Invasive tumor cells were found in 70% of on-treatment biopsies, with varying rates across subtypes (HR+/HER2−: 84%, TNBC: 62%, HER2+: 51%; p < 0.001). Patients with residual tumor on-treatment had an 8% pCR rate post-treatment (HR+/HER2−: 3%, TNBC: 19%, HER2+: 11%), while those without any invasive tumor had a 50% pCR rate (HR+/HER2−: 27%; TNBC: 48%, HER2+: 66%). Sensitivity for predicting residual disease was 0.81, with positive and negative predictive values of 0.92 and 0.50, respectively. Increasing TILs from baseline to on-treatment biopsy (if residual tumor was present) were linked to higher pCR likelihood in the overall cohort (OR 1.034, 95% CI 1.013–1.056 per % increase; p = 0.001) and with a longer DFS in TNBC (HR 0.980, 95% CI 0.963–0.997 per % increase; p = 0.026). Persisting or increased Ki-67 was associated with with lower pCR probability in the overall cohort (OR 0.957, 95% CI 0.928–0.986; p = 0.004) and shorter DFS in TNBC (HR 1.023, 95% CI 1.001–1.047; p = 0.04).ConclusionOn-treatment biopsies can predict patients unlikely to achieve pCR post-therapy. This could facilitate therapy adjustments for TNBC or HER2 + BC. They also might offer insights into therapy resistance mechanisms. Future research should explore whether standardized or expanded sampling enhances the accuracy of on-treatment biopsy procedures.Trial registration GeparQuattro (EudraCT 2005-001546-17), GeparQuinto (EudraCT 2006-005834-19) and GeparSixto (EudraCT 2011-000553-23).

Features of beta cell differentiation during the development of type 2 diabetes mellitus

Type 2 diabetes mellitus is characterized by a mild inflammatory reaction in the pancreas, which affects the structure and function of the pancreatic islets: the number of β-cells decreases and the number of α-cells increases. The work examined the features of β-cell differentiation in the development of experimental type 2 diabetes mellitus and while reducing the inflammatory process. Biochemical, histological methods, enzyme-linked immunosorbent assay, immunohistochemical methods were used using primary antibodies to insulin, glucagon, proliferation marker Ki-67 and secondary antibodies labeled with fluorescent dyes. Streptozotocin and nicotinamide were used to model type 2 diabetes mellitus, and the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione was used to reduce the inflammatory response. Previous studies have shown that it changes the macrophage phenotype from proinflammatory M1 to anti-inflammatory M2. In type 2 diabetes mellitus, against the background of a decrease in the number of macrophages with the CD163 marker and the concentration of the cytokine TGF-β1, which have an anti-inflammatory effect, in the pancreatic islets, a decrease in the number of β-cells and their functional activity was observed, while the content of α-cells synthesizing glucagon increased. After administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, the opposite picture was observed in the pancreatic islets: against the background of an increase in the number of CD163+ macrophages and the content of TGF-β1, the number of β cells increased and the number of α cells decreased-cells. The increase in the number of insulin-synthesizing cells was not accompanied by their mitotic activity. It is likely that a decrease in the number of CD163+ macrophages and the level of the antiinflammatory cytokine TGF-β1 in the islets are factors contributing to changes in the cell microenvironment and, as a consequence, the differentiation of β-cells into α-cells. On the contrary, an increase in the number of CD163+ macrophages and TGF-β1 against the background of administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione presumably promotes reverse differentiation of α-cells into β-cells and restoration of insulin synthesis pancreas. Targeted effects on the microenvironment of cells in the pancreatic islet in type 2 diabetes mellitus may be a new approach to treating the disease.

α-Parvin Expression in Breast Cancer Tissues: Correlation with Clinical Parameters and Prognostic Significance.

Stromal cells play a critical role in the tumor microenvironment of breast cancer (BC), as they are recruited by tumor cells and regulate the metastatic spread. Though high expression of α-parvin, a member of the parvin family of actin-binding proteins, is reported to be associated with a poor prognosis and metastasis in several cancers, its role in carcinogenesis has not been thoroughly explored. Therefore, we aimed to examine the expression of α-parvin in BC patients by compartmentalizing and quantifying tissues to determine whether α-parvin can be a potential therapeutic target. We performed immunohistochemical (IHC) staining of α-parvin in BC tissues, and the IHC scores were calculated in the overall tissue, stroma, and epithelium using image analysis software. The expression of α-parvin was significantly higher in BC tissues (p = 0.0002) and BC stroma (p < 0.0001) than in normal tissues. Furthermore, all α-parvin scores were significantly positively correlated with the proliferation marker Ki67. The overall and stroma scores are associated with the tumor, (lymph) node, and metastasis (TNM) classification, stage, and grade. These results suggest that high expression of α-parvin in stroma is associated with BCs and might be a new predictive marker for diagnosing BC.

Doublecortin-immunoreactive neurons in the piriform cortex are sensitive to the long lasting effects of early life stress.

The olfactory system is a niche of continuous structural plasticity, holding postnatal proliferative neurogenesis in the olfactory bulbs and a population of immature neurons in the piriform cortex. These neurons in the piriform cortex are generated during embryonic development, retain the expression of immaturity markers such as doublecortin, and slowly mature and integrate into the olfactory circuit as the animal ages. To study how early life experiences affect this population of cortical immature neurons, we submitted mice of the C57/Bl6J strain to a protocol of maternal separation for 3 h per day from postnatal day 3 to postnatal day 21. Control mice were continuously with their mothers. After weaning, mice were undisturbed until 6 weeks of age, when they were weighted and tested in the elevated plus-maze, a standard test for anxiety-like behavior, to check for phenotypical effects. Mice were then perfused, and their brains processed for the immunofluorescent detection of doublecortin and the endogenous proliferation marker Ki67. We found that maternal separation induced a significant increase in the body weight of males, but not females. Further, maternally separated mice displayed increased exploratory-like behavior (i.e., head dipping, velocity and total distance traveled in the elevated plus maze), but no significant differences in anxiety-like behavior or corticosterone levels after behavioral testing. Finally, we observed a significant increase in the number of complex doublecortin neurons in the piriform cortex, but not in the olfactory bulbs, of mice submitted to maternal separation. Interestingly, most doublecortin neurons in the piriform cortex, but not the olfactory bulb, express the epigenetic reader MeCP2. In summary, mild early life stress results, during adolescence, in a male-specific increase in body weight, alteration of the exploratory behaviors, and an increase in doublecortin neurons in the piriform cortex, suggesting an alteration in their maturation process.

Human intermediate prostate cancer stem cells contribute to the initiation and development of prostate adenocarcinoma

BackgroundIntermediate cells are present in the early stages of human prostate development and adenocarcinoma. While primary cells isolated from benign human prostate tissues or tumors exhibit an intermediate phenotype in vitro, they cannot form tumors in vivo unless genetically modified. It is unclear about the stem cell properties and tumorigenicity of intermediate cells.MethodsWe developed a customized medium to culture primary human intermediate prostate cells, which were transplanted into male immunodeficient NCG mice to examine tumorigenicity in vivo. We treated the cells with different concentrations of dihydrotestosterone (DHT) and enzalutamide in vitro and surgically castrated the mice after cell transplantation in vivo. Immunostaining, qRT-PCR, RNA sequencing, and western blotting were performed to characterize the cells in tissues and 2D and 3D cultures.ResultsWe found intermediate cells expressing AR+PSA+CK8+CK5+ in the luminal compartment of human prostate adenocarcinoma by immunostaining. We cultured the primary intermediate cells in vitro, which expressed luminal (AR+PSA+CK8+CK18+), basal (CK5+P63+), intermediate (IVL+), and stem cell (CK4+CK13+PSCA+SOX2+) markers. These cells resisted castration in vitro by upregulating the expression of AR, PSA, and proliferation markers KI67 and PCNA. The intermediate cells had high tumorigenicity in vivo, forming tumors in immunodeficient NCG mice in a month without any genetic modification or co-transplantation with embryonic urogenital sinus mesenchyme (UGSM) cells. We named these cells human castration-resistant intermediate prostate cancer stem cells or CriPCSCs and defined the xenograft model as patient primary cell-derived xenograft (PrDX). Human CriPCSCs resisted castration in vitro and in vivo by upregulating AR expression. Furthermore, human CriPCSCs differentiated into amplifying adenocarcinoma cells of luminal phenotype in PrDX tumors in vivo, which can dedifferentiate into CriPCSCs in vitro.ConclusionsOur study identified and established methods for culturing human CriPCSCs, which had high tumorigenicity in vivo without any genetic modification or UGSM co-transplantation. Human CriPCSCs differentiated into amplifying adenocarcinoma cells of luminal phenotype in the fast-growing tumors in vivo, which hold the potential to dedifferentiate into intermediate stem cells. These cells resisted castration by upregulating AR expression. The human CriPCSC and PrDX methods hold significant potential for advancing prostate cancer research and precision medicine.

Evaluation of epithelial-to-mesenchymal transition and Ki-67 index in aggressive papillary thyroid cancer

ObjectivesThe present study aims to characterize immunohistochemical features of markers associated with Epithelial–Mesenchymal Transition (EMT) and proliferative activity that could lead to death in Papillary Thyroid Cancer (PTC). MethodsClinical data and tumor material were retrospectively collected. The patients were separated into death from PTC (Group 1), metastatic cases with indolent behavior (Group 2) and non-metastatic indolent PTC (Group 3). Immunohistochemical assessment of E-cadherin, β-catenin, Vimentin, ZEB-1 and Ki-67 was performed in each tumor and a semiquantitative estimation of the percentage of expression was fulfilled on the best marking area at high of the tumor invasion front. Results31 patients were included, 15 that died from PTC (Group 1), 6 in Group 2 and 10 in Group 3. The proliferative marker Ki-67 showed a significant difference in its expression in the tumor invasion front between the groups, specifically between groups 1 and 3 (p = 0.006). On the other hand, EMT-related immunohistochemical markers did not show significant difference in their percentage of expression, since loss of E-cadherin, β-catenin and Vimentin was observed in most cases at the invasion front. ConclusionPatients that died from PTC had a significantly higher Ki-67 labelling index compared to patients with indolent disease (cutoff of 11%). Ki-67 may have a role as a prognostic marker and could be considered for routine use in PTC. Level of evidence4.

Synergistic anti-tumorigenic effect of diosmetin in combination with 5-fluorouracil on human colon cancer xenografts in nude mice

5-Fluorouracil (5-FU) is frequently used to treat colorectal cancer (CRC), but its clinical application is limited by its toxicity. Natural compounds have been combined with chemotherapeutic drugs to reduce chemotherapy-related toxicity. Diosmetin, a natural flavonoid, has demonstrated anticancer effects against CRC. This study investigated diosmetin's potential in combination with 5-FU using a murine model of HCT-116 colon cancer xenografts in nu/nu nude mice. HCT-116 cells were injected into the right flanks of mice, and once tumors reached a size of 50 mm3, the mice were treated with diosmetin (100 mg/kg), 5-FU (30 mg/kg), or a combination of both at two dose levels (100 + 30 mg/kg and 50 + 15 mg/kg) for 4 weeks. Blood and tumors were collected on the final day for further analysis. Mice treated with the higher combination dose exhibited the smallest tumor volume (330.91 ± 88.49 mm3). Biochemistry and histology analysis showed no toxicity or abnormalities in the liver, kidney, and heart with the combination therapy. Immunohistochemistry results revealed a notable reduction in the proliferation marker (Ki67) and inflammation marker (TLR4) in tumors from high-dose combination-treated mice. Moreover, immunofluorescence data indicated increased levels of apoptotic markers (Bax, Caspase-3, p53, p21) and downregulation of anti-apoptotic protein (Bcl-2) in the high-dose combination group. The findings suggest that 100 mg/kg of diosmetin combined with 30 mg/kg 5-FU significantly reduced tumor volume and had a less toxic effect on the heart compared to 5-FU monotherapy.

The oxytocin-oxytocin receptors system — a new pathogenetic mechanism in the development of diabetic phenotype of heart failure with preserved ejection fraction in women

Aim. To determine the pathogenetic role of the oxytocinergic system in the development of myocardium structural and functional changes in women with heart failure with preserved ejection fraction (HFpEF) associated with type 2 diabetes mellitus (DM2T) (diabetic phenotype of HFpEF).Material and methods. The study included 60 women aged 67.0±4.9 years with HFpEF stage I-IIA, FC I-III, 30 of them had DM2T who were admitted for elective coronary artery bypass grafting. The development of HFpEF is caused by coronary artery disease (CAD) and arterial hypertension (AH). Prior to surgery, all patients underwent a standard examination, blood levels of NT-proBNP, oxytocin (Ox), echocardiography were determined to find the types of left ventricular (LV) myocardial remodeling and diastolic dysfunction (DD). Myocardium biopsies of the right atrium auricle obtained during coronary bypass surgery were studied by microscopy, morphometry and immunohistochemistry (the expression of oxytocin receptors (OxR), a marker of proliferation ki-67).Results. According to echocardiography, eccentric LV hypertrophy (46.7/36.7%) and DD type 2 (47/17%, p=0.003) prevailed in the group of women with the diabetic phenotype of HFpEF. A higher content of NT-proBNP (480.72±241.87/434.46±282.78 ng/ml, p=0.06) and a lower concentration of Ox (102.11±35.89/320.37±294.71 pg/ml, p=0.0016) in blood serum were established, as well as an increase in the number of cardiomyocytes (CMC) with a high expression level OxR (63.69±19.47/12.16±23.09%, p=0.000) in patients with the diabetic phenotype of HFpEF. Negative associations were determined between the blood level of Ox and the CMC diameter (r=-0.10, p=0.020), the area of their cytoplasm (r=-0.16, p=0.000) and the area of the nuclei (r=-0.11, p=0.015) in patients of both groups. A decrease in Ox concentration in the blood of patients with diabetic phenotype of HFpEF was accompanied by an increase in the number of CMCs with a high level of OxR expression (r=-0.63, p=0.000).Conclusion. The study has shown the important involvement of oxytocinergic signaling pathways in the HFpEF pathogenesis. HFpEF associated with DM2T in women was characterized by more unfavorable structural and functional changes in the myocardium, a significant increase in the number of hypertrophied CMCs with a high level of OxR expression and Ox decrease in blood serum. The mechanisms of the first-established significant increase in the content of Ox in the blood of patients with HFpEF without diabetes and its significant decrease in patients with diabetic phenotype of HFpEF leading to more pronounced structural and functional changes in the myocardium, require further study.

Bio-Pathological Functions of Posttranslational Modifications of Histological Biomarkers in Breast Cancer.

Proteins are the most common types of biomarkers used in breast cancer (BC) theranostics and management. By definition, a biomarker must be a relevant, objective, stable, and quantifiable biomolecule or other parameter, but proteins are known to exhibit the most variate and profound structural and functional variation. Thus, the proteome is highly dynamic and permanently reshaped and readapted, according to changing microenvironments, to maintain the local cell and tissue homeostasis. It is known that protein posttranslational modifications (PTMs) can affect all aspects of protein function. In this review, we focused our analysis on the different types of PTMs of histological biomarkers in BC. Thus, we analyzed the most common PTMs, including phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, palmitoylation, myristoylation, and glycosylation/sialylation/fucosylation of transcription factors, proliferation marker Ki-67, plasma membrane proteins, and histone modifications. Most of these PTMs occur in the presence of cellular stress. We emphasized that these PTMs interfere with these biomarkers maintenance, turnover and lifespan, nuclear or subcellular localization, structure and function, stabilization or inactivation, initiation or silencing of genomic and non-genomic pathways, including transcriptional activities or signaling pathways, mitosis, proteostasis, cell-cell and cell-extracellular matrix (ECM) interactions, membrane trafficking, and PPIs. Moreover, PTMs of these biomarkers orchestrate all hallmark pathways that are dysregulated in BC, playing both pro- and/or antitumoral and context-specific roles in DNA damage, repair and genomic stability, inactivation/activation of tumor-suppressor genes and oncogenes, phenotypic plasticity, epigenetic regulation of gene expression and non-mutational reprogramming, proliferative signaling, endocytosis, cell death, dysregulated TME, invasion and metastasis, including epithelial-mesenchymal/mesenchymal-epithelial transition (EMT/MET), and resistance to therapy or reversal of multidrug therapy resistance. PTMs occur in the nucleus but also at the plasma membrane and cytoplasmic level and induce biomarker translocation with opposite effects. Analysis of protein PTMs allows for the discovery and validation of new biomarkers in BC, mainly for early diagnosis, like extracellular vesicle glycosylation, which may be considered as a potential source of circulating cancer biomarkers.