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Ki-67 expression in non-cancerous and cancerous cells upon serum starvation Confocal analysis of Ki-67 expression in HDF, HUVEC, MDA-MB-231, HeLa and FaDu cells (analysed over time after serum deprivation), and the relative fluorescence signal quantification (average overlap value percent). (Two-way ANOVA, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Scale bar: 10 μm.
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Ki-67 is a nuclear protein that has been used in cancer diagnostic because of its specific cell-cycle dependent expression profile. After quantifying and characterising the expression level of Ki-67, as a function of the cell cycle, we found out that the two main splice variants of the protein (i.e. α and β) are differently regulated in non-cancero...
Citations
... To strengthen the observations of the dual inhibitors towards the cell cycle mediated by cyclin D2 overexpression, we further assessed the cell proliferation status. To this end, we analyzed the temporal expression of Ki-67, a key player involved in tumor cell proliferation [47], in the presence of molecules 25 or 27 at their IC 50 s concentration (Figure 7). Regarding the effects of the molecules in cyclin D2 immunoreactivity, we observed that untreated cells presented low expression of this regulator of G1-S transition, while with both molecules' treatment, an increase was observed ( Figure 6A). ...
... To strengthen the observations of the dual inhibitors towards the cell cycle mediated by cyclin D2 overexpression, we further assessed the cell proliferation status. To this end, we analyzed the temporal expression of Ki-67, a key player involved in tumor cell proliferation [47], in the presence of molecules 25 or 27 at their IC50′s concentration (Figure 7). Semi-quantitative analysis of Ki-67 nuclear expression (B) revealed a significant decrease of Ki-67 nuclear immunoreactivity after incubation with mol 25 or 27 from 3 and 6 h onwards, respectively. ...
Triple-negative breast cancer (TNBC) is a devastating BC subtype. Its aggressiveness, allied to the lack of well-defined molecular targets, usually culminates in the appearance of metastases that account for poor prognosis, particularly when they develop in the brain. Nevertheless, TNBC has been associated with epidermal growth factor receptor (EGFR) overexpression, leading to downstream phosphoinositide 3-kinase (PI3K) signaling activation. We aimed to unravel novel drug candidates for TNBC treatment based on EGFR and/or PI3K inhibition. Using a highly metastatic TNBC cell line with brain tropism (MDA-MB-231 Br4) and a library of 27 drug candidates in silico predicted to inhibit EGFR, PI3K, or EGFR plus PI3K, and to cross the blood–brain barrier, we evaluated the effects on cell viability. The half maximal inhibitory concentration (IC50) of the most cytotoxic ones was established, and cell cycle and death, as well as migration and EGFR pathway intervenient, were further evaluated. Two dual inhibitors emerged as the most promising drugs, with the ability to modulate cell cycle, death, migration and proliferation, morphology, and PI3K/AKT cascade players such as myocyte enhancer factor 2C (MEF2C) and forkhead box P1 (FOXP1). This work revealed EGFR/PI3K dual inhibitors as strong candidates to tackle brain metastatic TNBC cells.
... [36] A recent study confirmed that specific MKI67 splice variants contribute to cancer progression by affecting the cell cycle. [37] MKI67 proliferation index can be used as a marker of recurrence time of intracranial meningiomas. [38] MKI67 is a nuclear protein expressed in all proliferating vertebrate cells and is a widely used biomarker to estimate the proportion of dividing cells to grade tumors. ...
Oral squamous cell carcinoma is a malignant tumor that occurs in the oral cavity, with poor prognosis and easy recurrence. However, the relationship between MKI67 and oral squamous cell carcinoma remains unclear. The oral squamous cell carcinoma datasets GSE138206, GSE146483 and GSE184616 were downloaded from the gene expression omnibus database, and the differentially expressed genes (DEGs) were screened. The protein-protein interaction network was constructed and analyzed by search tool for the retrieval of interacting genes database and Cytoscape software. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) were used for functional enrichment analysis. GO and KEGG analyses were performed on the whole genome, as formulated by gene set enrichment analysis. comparative toxicogenomics database was used to identify the diseases most associated with the core genes. TargetScan was used to screen miRNA regulating central DEGs. A total of 1472 DEGs were identified. GO analysis showed that the differentially expressed genes were mainly enriched in the tissues of extracellular matrix, type i interferon signaling pathway, human papillomavirus infection, adhesion spot, hepatitis C and ECM-receptor interaction. Enrichment items were similar to GO and KEGG enrichment items of differentially expressed genes. 10 core genes were obtained, and their expression was different between oral squamous cell carcinoma and normal tissue samples. MKI67 is highly expressed in oral squamous cell carcinoma and may be an oncogene in oral squamous cell carcinoma.
... However, recent findings indicate that Ki-67 should not be considered only as a marker of cell proliferation [88,89]. Since differences in the extranuclear pathway of Ki-67 regulation in non-cancer and cancer cells have been identified [90], additional studies will potentially provide answers related to the biological role of nuclear and cytoplasmic Ki67 in adult stem cells. ...
The heterogeneity of stem cells represents the main challenge in regenerative medicine development. This issue is particularly pronounced when it comes to the use of primary mesenchymal stem/stromal cells (MSCs) due to a lack of identification markers. Considering the need for additional approaches in MSCs characterization, we applied Raman spectroscopy to investigate inter-individual differences between bone marrow MSCs (BM-MSCs). Based on standard biological tests, BM-MSCs of analyzed donors fulfill all conditions for their characterization, while no donor-related specifics were observed in terms of BM-MSCs morphology, phenotype, multilineage differentiation potential, colony-forming capacity, expression of pluripotency-associated markers or proliferative capacity. However, examination of BM-MSCs at a single-cell level by Raman spectroscopy revealed that despite similar biochemical background, fine differences in the Raman spectra of BM-MSCs of each donor can be detected. After extensive principal component analysis (PCA) of Raman spectra, our study revealed the possibility of this method to diversify BM-MSCs populations, whereby the grouping of cell populations was most prominent when cell populations were analyzed in pairs. These results indicate that Raman spectroscopy, as a label-free assay, could have a huge potential in understanding stem cell heterogeneity and sorting cell populations with a similar biochemical background that can be significant for the development of personalized therapy approaches.
... Herein, we observed a reduction in the Ki-67-positive cells after exposure to compound 3q for 24 and 48 h, confirming the results obtained by the trypan blue assay. The cell proliferation marker Ki-67, a nuclear protein, is widely used in cancer diagnosis because its expression is dependent on the phases of the cell cycle [66]. Additionally, we decided to analyze the cell cycle once several cell cycle regulators have emerged as potential therapeutic drugs for cancer treatment [67]. ...
Melanoma is the most aggressive skin cancer, and its incidence has continued to rise during the past decades.
Conventional treatments present severe side effects in cancer patients, and melanoma can be refractory to
commonly used anticancer drugs, which justify the efforts to find new potential anti-melanoma drugs. An
alternative to promote the discovery of new pharmacological substances would be modifying chemical groups
from a bioactive compound. Here we describe the synthesis of seventeen compounds derived from cinnamic acid
and their bioactivity evaluation against melanoma cells. The compound phenyl 2,3-dibromo-3-phenylpropanoate
(3q) was the most effective against murine B16-F10 cells, as observed in cytotoxicity and cell migration assays.
Simultaneously, this compound showed low cytotoxic activity on non-tumor cells. At the highest concentration,
the compound 3q was able to trigger apoptosis, whereas, at lower concentrations, it affected the cell cycle and
melanoma cell proliferation. Furthermore, cinnamate 3q impaired cell invasion, adhesion, colonization, and
actin polymerization. In conclusion, these results highlight the antiproliferative and antimetastatic potential of
cinnamic acid derivatives on melanoma.
... For example, the most pronounced exon exclusion event was found in the gene MKI67, which encodes a cell proliferation marker, Ki-67, commonly used in prognostic evaluation of prostate and other cancers. Ki-67 exists in cells in two predominant splice isoforms distinguished by the inclusion or exclusion of exon 7, the longer of which is associated with proliferating cancer cells (Chierico et al., 2017). PRMT1 knockdown in LNCaP cells promotes exclusion of MKI67 exon 7, leading to increased expression of the short isoform (MKI67-S) and ...
Androgen receptor (AR) signaling is the central driver of prostate cancer across disease states. While androgen deprivation therapy (ADT) is effective in the initial treatment of prostate cancer, resistance to ADT or to next-generation androgen pathway inhibitors invariably arises, most commonly through the re-activation of the AR axis. Thus, orthogonal approaches to inhibit AR signaling in advanced prostate cancer are essential. Here, via genome-scale CRISPR-Cas9 screening, we identify protein arginine methyltransferase 1 (PRMT1) as a critical mediator of AR expression and signaling. PRMT1 regulates the recruitment of AR to genomic target sites and the inhibition of PRMT1 impairs AR binding at lineage-specific enhancers, leading to decreased expression of key oncogenes, including AR itself. In addition, AR-driven prostate cancer cells are uniquely susceptible to combined AR and PRMT1 inhibition. Our findings implicate PRMT1 as a key regulator of AR output and provide a preclinical framework for co-targeting of AR and PRMT1 in advanced prostate cancer.
... Although MKI67 RNA expression was not upregulated in our cohort, i.e., the transcript encoding the Ki-67 protein, we decided to use Ki-67 as a marker for T cell proliferation. Ki-67 is an established proliferation marker and the absence of MKI67 upregulation might be explained by the fact that Ki-67 protein expression is partly regulated by proteasomal degradation [30]. We were able to detect an increase in Ki-67 + CD8 + T cells in patients with clinical benefit, supporting the idea that proliferating T cells are accountable for the observed changes in DNA replication/cell cycle genes. ...
Although immune checkpoint inhibitors improve median overall survival in patients with metastatic urothelial cancer (mUC), only a minority of patients benefit from it. Early blood-based response biomarkers may provide a reliable way to assess response weeks before imaging is available, enabling an early switch to other therapies. We conducted an exploratory study aimed at the identification of early markers of response to anti-PD-1 in patients with mUC. Whole blood RNA sequencing and phenotyping of peripheral blood mononuclear cells were performed on samples of 26 patients obtained before and after 2 to 6 weeks of anti-PD-1. Between baseline and on-treatment samples of patients with clinical benefit, 51 differentially expressed genes (DEGs) were identified, of which 37 were upregulated during treatment. Among the upregulated genes was PDCD1, the gene encoding PD-1. STRING network analysis revealed a cluster of five interconnected DEGs which were all involved in DNA replication or cell cycle regulation. We hypothesized that the upregulation of DNA replication/cell cycle genes is a result of T cell proliferation and we were able to detect an increase in Ki-67+ CD8+ T cells in patients with clinical benefit (median increase: 1.65%, range −0.63 to 7.06%, p = 0.012). In patients without clinical benefit, no DEGs were identified and no increase in Ki-67+ CD8+ T cells was observed. In conclusion, whole blood transcriptome profiling identified early changes in DNA replication and cell cycle regulation genes as markers of clinical benefit to anti-PD-1 in patients with urothelial cancer. Although promising, our findings require further validation before implementation in the clinic.
... Ki-67 expression is an independent prognostic parameter according to breast cancer molecular subtypes in breast cancer patients [48]. At the molecular level, expression, localization and post-translational modification of Ki-67 are regulated by the cell cycle [49]; during the early G1 phase, the Ki-67 antigen is located in the nucleoplasm, unlike its nucleolus location in the S and G2 phases [50], and is regulated differently in non-cancerous and cancerous cells [51], providing an insight into the mechanism of CAP selectivity towards cancerous cells. Our data on Ki-67 expression in four breast cancer subtypes indicate down-regulation of Ki-67 by CAP with slightly different dosages. ...
Breast cancer is the most common cancer among women worldwide. Its molecular receptor marker status and mutational subtypes complicate clinical therapies. Cold atmospheric plasma is a promising adjuvant therapy to selectively combat many cancers, including breast cancer, but not normal tissue; however, the underlying mechanisms remain unexplored. Here, four breast cancer cell lines with different marker status were treated with Canady Helios Cold Plasma™ (CHCP) at various dosages and their differential progress of apoptosis was monitored. Inhibition of cell proliferation, induction of apoptosis, and disruption of the cell cycle were observed. At least 16 histone mRNA types were oxidized and degraded immediately after CHCP treatment by 8-oxoguanine (8-oxoG) modification. The expression of DNA damage response genes was up-regulated 12 h post-treatment, indicating that 8-oxoG modification and degradation of histone mRNA during the early S phase of the cell cycle, rather than DNA damage, is the primary cause of cancer cell death induced by CHCP. Our report demonstrates for the first time that CHCP effectively induces cell death in breast cancer regardless of subtyping, through histone mRNA oxidation and degradation during the early S phase of the cell cycle.
... Nonetheless, the sharp gene expression change observed between G3 and G2, even in matched samples from the same patient, suggests that further investigation in this direction may provide new clues on the malignant progression of PanNETs, and possibly on how to interfere with it. In fact, in addition to the refined role in chromosome dynamics during mitosis, Ki67 has multiple molecular functions that display cell-type-specific variations [29,30]. ...
Simple Summary
Ki67-based grading is a major prognostic parameter for pancreatic neuroendocrine tumors. Gene expression profiles of these tumors have been explored, yet their relationship with Ki67-based tumor grade has only been superficially investigated. To fill this gap, we analyzed differentially expressed genes across 29 cases of different grades. Our data provided the first proof that the switch from lower to higher grades is associated with a profound change in the transcriptome. The comparison of multiple samples from the same patients, including primaries and metastasis, showed that the major determinant of difference was tumor grade, irrespective of the anatomic location or patient of origin. These data call for further investigation of this association and of the role of Ki67 in affecting chromosomal stability in neuroendocrine tumors of different grades, which may clarify the basis of tumor progression and provide clues on how to interfere with it.
Abstract
Pancreatic neuroendocrine tumors (PanNETs) display variable aggressive behavior. A major predictor of survival is tumor grade based on the Ki67 proliferation index. As information on transcriptomic profiles of PanNETs with different tumor grades is limited, we investigated 29 PanNETs (17 G1, 7 G2, 5 G3) for their expression profiles, mutations in 16 PanNET relevant genes and LINE-1 DNA methylation profiles. A total of 3050 genes were differentially expressed between tumors with different grades (p < 0.05): 1279 in G3 vs. G2; 2757 in G3 vs. G1; and 203 in G2 vs. G1. Mutational analysis showed 57 alterations in 11 genes, the most frequent being MEN1 (18/29), DAXX (7/29), ATRX (6/29) and MUTYH (5/29). The presence and type of mutations did not correlate with the specific expression profiles associated with different grades. LINE-1 showed significantly lower methylation in G2/G3 versus G1 tumors (p = 0.007). The expression profiles of matched primaries and metastasis (nodal, hepatic and colorectal wall) of three cases confirmed the role of Ki67 in defining specific expression profiles, which clustered according to tumor grades, independently from anatomic location or patient of origin. Such data call for future exploration of the role of Ki67 in tumor progression, given its involvement in chromosomal stability.
... However, a pathway was recently discovered for eliminating extranuclear Ki-67, through which it is transported to the Golgi apparatus. (4) It was shown that in normal cells Ki-67 is a late marker of entry into the cell cycle; Ki-67 mRNA fluctuated with maximum levels in G2, while Ki-67 protein levels increased throughout the cell cycle, peaking in mitosis. After exit from the cell cycle, Ki-67 expression remains at a low level, but is not detected in uncycling differentiated cells or senescent cells. ...
... In Group 2, there was no significant increase in the severity of the adhesive process; by Day 30 it was estimated at 4 points ( Figure 2). Differences in the severity of the adhesive process between the groups are significant on Days 7, 14 and 30: on Day 7 in Group 1, the score was 9 [8][9][10][11][12] points, in points (P=0.0159); on Day 14 -9[6-9] and 3 [3][4] points, respectively (P=0.0159). The differences between the groups increased to Day 30 -up to 12 [9][10][11][12][13] and 4 points (P=0.0079). ...
Background: Ki-67 is a nuclear protein expressed in all proliferating cells of vertebrates during mitotic cycle phases S, G1, G2, and M, except for G0. Studying this marker is widely used to diagnose the proliferative activity of tumors. However, studying Ki-67 in non-neoplastic diseases attracts much less attention among the researchers. The aim of this study was to assess the possibility of using staining for Ki-67 to identify the proliferative potential of fibroblasts during the formation of adhesions in the abdominal cavity (AC). Methods and Results: Experiments were carried out on male Wistar rats. The adhesion process in AC was simulated in the control group (n=25), and in the experimental group (n=25) with the administration of Seroguard®. Animals were sacrificed on Days 1–30, and the severity of the adhesive process in AC was assessed. Histological sections were prepared and stained for Ki-67. It was found that the animals of the control group had increased severity of the adhesive process in AC during the observation. Maximum increase in severity was registered on Day 30 – 12[9-13] points in the control group and 4[4-4] points in the experimental group (P=0.0079). High proliferative activity of fibroblasts in the control group was detected on Days 3, 7, 14 and 30, which may indicate an active division of fibroblasts and the formation of adhesions in the damaged area. In the experimental group, single Ki-67 positive cells were noted during the entire observation period, which may point to a reduced potential for the formation of adhesions. Conclusion: Our study showed the prospects of using Ki-67 staining to determine the severity of the developing adhesive process in AC, and also revealed one of the possible mechanisms that inhibit the formation of the adhesive process when using Seroguard® – a decrease in the mitotic activity of fibroblasts in the area of peritoneal injury.
... It is made available under a The copyright holder for this preprint this version posted June 17, 2020. . https://doi.org/10.1101/2020.06.17.156034 doi: bioRxiv preprint longer of which is associated with proliferating cancer cells (60). PRMT1 knockdown in LNCaP cells promotes exclusion of MKI67 exon 7, leading to increased expression of the short isoform (MKI67-S) and decreased expression of the pro-proliferative long isoform (MKI67-L) (Fig. S4 C and D). ...
Androgen receptor (AR) signaling is the central driver of prostate cancer growth and progression across disease states, including in most cases of castration-resistant prostate cancer (CRPC). While next-generation AR antagonists and androgen synthesis inhibitors are effective for a time in CRPC, tumors invariably develop resistance to these agents, commonly through mechanisms resulting in the overexpression of AR or the production of constitutively active AR splice variants (e.g. AR-V7). Improved mechanistic understanding of the factors that modulate AR expression and signaling may reveal additional therapeutic intervention points in CRPC. Here, we leverage genome-scale CRISPR/Cas9 genetic screening to systematically identify regulators of AR/AR-V7 expression. We identify protein arginine methyltransferase 1 (PRMT1) as a critical mediator of AR expression and signaling that regulates recruitment of AR to genomic target sites. PRMT1 suppression globally perturbs the expression and splicing of AR target genes and inhibits the proliferation and survival of AR-positive prostate cancer cells. Genetic or pharmacologic inhibition of PRMT1 reduces AR binding at lineage-specific enhancers, which leads to decreased expression of key oncogenes, including AR itself. CRPC cells displaying activated AR signaling due to overexpression of AR or AR-V7 are uniquely susceptible to combined AR and PRMT1 inhibition. Our findings implicate PRMT1 as a critical regulator of AR output and provide a preclinical framework for co-targeting of AR and PRMT1 as a promising new therapeutic strategy in CRPC.
