Nucleus Of Cancer Cells Research Articles

Single-Cell Analysis with Silver-Coated Pipette by Combined SERS and SICM.

The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for the cell analysis by combination with surface-enhanced Raman spectroscopy (SERS) and scanning ion-conducting microscopy (SICM). Unlike the majority of other designs, the pipette opening in our case remains uncovered, which is important for SICM. SERS-active Ag nanoparticles on the pipette surface are formed by vacuum-thermal evaporation followed by annealing. An array of nanoparticles had a diameter on the order of 36 nm and spacing of 12 nm. A two-particle model based on Laplace equations is used to calculate a theoretical enhancement factor (EF). The surface morphology of the samples is investigated by scanning electron microscopy while SICM is used to reveal the surface topography, to evaluate Young's modulus of living cells and to control an injection of the SERS-active pipettes into them. A Raman microscope-spectrometer was used to collect characteristic SERS spectra of cells and cell components. Local Raman spectra were obtained from the cytoplasm and nucleus of the same HEK-293 cancer cell. The EF of the SERS-active pipette was 7 × 105. As a result, we demonstrate utilizing the silver-coated pipette for both the SICM study and the molecular composition analysis of cytoplasm and the nucleus of living cells by SERS. The probe localization in cells is successfully achieved.

Pr3+ Ion-Substituted Ni-Co Nano-Spinel Ferrites: Their Synthesis, Characterization, and Biocompatibility for Colorectal Cancer and Candidaemia.

Nanotherapeutics have attracted tremendous research interest in the modern pharmaceutical and biomedical industries due to their potential for drug development, targeted delivery, and therapeutic applications. Therefore, the current study underpins the synthesis of praseodymium ion (Pr3+)-substituted Ni0.5Co0.5Fe2O4 nano-spinel ferrites, (Co0.5Ni0.5PrxFe2-xO4 (0.0 ≤ x ≤ 0.10) NSFs, CoNiPr (x ≤ 0.10) NSFs) via the sonochemical route for its application as a nanotherapeutic treatment option. The synthesized nanomaterial was characterized using various analytical techniques, including scanning/transmission electron microscopy (SEM) and X-ray powder diffractometry (XRD). After substitution with Pr (x = 0.08), the particle size, polydispersity index, and zeta potential analysis indicated an increase in hydrodynamic diameter, with an average zeta potential value of -10.2 mV. The investigation of CoNiPr (x ≤ 0.10) NSFs on colorectal cancer (HCT-116) cells demonstrated a significant effect on cancer cell viability. The inhibitory concentration (IC50) of CoNiPr (x ≤ 0.10) NSFs was between 46 ± 0.91 and 288 ± 8.21 for HCT-116 cells. The effect of CoNiPr (x ≤ 0.10) NSFs on normal human embryonic kidney (HEK-293) cells showed a reduction in the HEK-293 cell viability; however, the cell viability was better than HCT-116. The NSFs treatment also showed morphological changes in cancer cell nuclei, as revealed by DAPI (4',6-diamidino-2-phenylindole), nuclear disintegration, and chromatic fragmentation, which are signs of apoptosis or programmed cell death. To examine the potential antifungal effects of CoNiPr NSFs on Candida albicans, known to cause candidemia among cancer patients, the viability of the cells was assessed post treatment with CoNiPr (x ≤ 0.10) NSFs. The increasing ratio of dopant had a moderate impact on the percentage of cell viability loss of 42, 44, and 43% with x = 0.06, 0.08, and 0.10, respectively. These results reinforce that increased dopant significantly impacts the antifungal properties of the synthesized nanomaterial. These findings support the idea that NSFs might be useful in pharmaceuticals.

Effect of cyclic substituents on the anti-cancer activity and DNA interaction of ruthenium(II) bis-phenanthroline dipyridoquinoline.

Introduction: Ruthenium(II) complexes have emerged recently as candidates for anti-cancer therapy, where activity is related to lipohilicity, cellular localization, and specific interactions with biomolecules. Methods: In this work, two novel complexes were synthesized and are reported based on the [Ru(phen)2(dipyrido[3,2-f:2',3'-h]quinoxaline]2+ framework. Results: Compared to the parent complex, annealing of cyclopenteno and cyclohexeno rings to the extended ligand substantially increased cytotoxicity towards a number of cancer cell lines, and induced apoptosis. The complexes localize in the nuclei of cancer cells and co-locate with DAPI on DNA. DNA binding studies show that both complexes bind strongly to DNA and one complex intercalates DNA like the parent, whilst the other appears to have multiple modes of interaction. Discussion: It is likely that the increased lipophilicity of the novel complexes is a key factor for increasing their cytotoxicity, rather than their DNA binding mode.

Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody

BackgroundPeptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer.MethodsThe subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes.ResultsPADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated.ConclusionsPADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.Graphical abstract

Phosphorylation of EZH2 differs HER2-positive breast cancer invasiveness in a site-specific manner

HER2-positive breast cancer (BC) invasiveness and drug-resistance issue is the critical treatment obstacle recently. We investigated the total and phosphorylated status EZH2 expression in database and BC tissue microarray. We demonstrated for the first time that EZH2 is distributed both in cytoplasm and nucleus of breast cancer cells in a phosphorylation site-specific manner. High expressed-EZH2 cases more frequently had an advanced clinical stage (lymph node metastasis) and aggressive features than EZH2-low cases, potentially indicating the high risk of HER2-positive BC (p < 0.05). Notably, highly expressed phosphorylated EZH2 is differently located in cytoplasm or nucleus in a site-specific manner in breast cancer cells. Nucleus-located pEZH2-S21 is expressed in invasive and lymph node metastatic HER2-positive BC cases (p = 0.144, p = 0.001). Cytoplasmic pEZH2-T487 is correlated with HER2 positive status (p = 0.014).In conclusion, high expression of nucleus-located EZH2 might be a predictor of invasive BC. Activation of phosphorylated EZH2-S21 site in nucleus would be a potential predictor of HER2-positve BC and poor efficacy of HER2-target therapy. These results point to a PRC2-independent non-epigenetic mechanism and therapeutic strategy of EZH2 in HER2-positive BC.

OR22-05 Progesterone Receptor-Grb2 Interaction Is Associated With Better Outcomes In Breast Cancer

Abstract Disclosure: N. Wittayavimol: None. E. Iwabuchi: None. P. Pateetin: None. Y. Miki: None. Y. Onodera: None. H. Sasano: None. V. Boonyaratanakornkit: None. The presence of progesterone receptor (PR) is usually associated with better clinical outcomes for several endocrine-related cancers. However, the molecular mechanism of how PR mediates growth inhibition remains unclear. In addition to its role as a nuclear transcription, PR also possesses an extranuclear function, mediated mainly through PR polyproline domain (PPD) interaction with the SH3 domains of cytoplasmic signaling molecules. Our previous study suggested a possible role of PR-PPD in mediating SH3-domain interaction. We showed that PR-PPD and SH3 domain interaction inhibited EGF-mediated signaling and decreased lung cancer cell proliferation. An essential adapter protein that helps transduce growth factor signaling is Grb2 which has an SH2 domain flanked by two SH3 domains. In this study, we hypothesized that PR interferes with cytoplasmic signaling through interaction between PR-PPD and Grb2-SH3. We used several protein-protein interaction assays in vitro and in cells to demonstrate PR-Grb2 interaction. GST-pulldown analysis showed that PR-PPD interacts specifically with SH3 domains of Grb2. No interaction was detected when key prolines in the PR-PPD to alanines. Immunofluorescence staining showed colocalization of PR and Grb2 in both the nucleus and cytoplasm of BT474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we showed that PR and Grb2 interacted in breast cancer cells, specifically through the Grb2-SH3 domain. No BiFC signal was detected when Grb2-SH3 domains were deleted. Furthermore, we used Proximity Ligation Assay (PLA) analysis to examine protein-protein interaction of a distance less than 40nm in cells. We found PR-Grb2 interaction in PR-positive breast cancer tissue sections. No PLA signal was detected in PR-negative breast cancer cells. Importantly, we examined 43 PR-positive breast cancer specimens and showed that PR-Grb2 interaction was associated with low histological stage and negatively correlated with lymph node invasion and metastasis of breast cancer. Our results provide a molecular mechanism for how PR can inhibit growth factor signaling by interfering with Grb2 signaling and a basis for developing novel therapeutic strategies for future cancer treatment. Presentation: Saturday, June 17, 2023

RAP80 Phase Separation at DNA Double-Strand Break Promotes BRCA1 Recruitment and Tumor Radio-Resistance

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 are not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, may be implicated. Recently, we and other groups have reported that liquid-liquid phase separation (LLPS) is a pivotal mechanism underlying DNA repair factors condensation at DSBs and their function. In this study, we aim to disclose whether RAP80 undergoes LLPS at DSBs and whether it is required for BRCA1 recruitment. To verify RAP80 is an LLPS protein and its function in DNA damage response (DDR): (1) candidate-mEGFP fusion protein formed condensates in cells and showed fluorescence recovery after photobleaching (FRAP); (2) candidate protein was expressed in Escherichia coli and purified with GST; (3) intrinsically disordered region (IDR) of RAP80 were predicted and tested in cell and in vitro; (4) lentivirus were used to construct RAP80-Knock out (KO) and RAP80 re-expression cell lines; (5) length gradient K63 poly-ubiquitin chains were chemically synthesized and incubated with RAP80 protein in vitro; (6) BRCA1 and RAP80 location were determined through immunofluorescence; (7) RAP80 protein expression in tissue was determined by IHC staining. Thin layer scanning and 3D reconstruction of the RAP80-mEGFP-expressing cells under a fluorescence microscope showed that RAP80-mEGFP formed spherical condensates with fast FRAP. Observation of purified proteins revealed that GST-RAP80-mEGFP protein formed liquid-like droplets, presenting as a FRAP and the fusion event among adjacent droplets. PEG-8000 and Ficol-400 strengthened the formation of GST-RAP80-mEGFP droplets in vitro. Later, we used a previously developed optoIDR tool to verify that IDR1 (1-254aa) is critical for RAP80 LLPS. To investigate whether the interaction between RAP80 and K63 poly-ubiquitin chains could enhance the condensation of RAP80, we chemically synthesized K63 ubiquitin chains and incubated them with purified GST-RAP80-mCherry proteins. The results showed that supplementation of ubiquitin multipolymer (poly-ubiquitin) significantly induced the LLPS of RAP80, and the ability of RAP80 condensates formation potency was positively correlated with the length of the ubiquitin chain. Consistent with their LLPS capacity, RAP80-WT-mEGFP, RAP80-(IDR1+AIR)-mEGFP groups showed prominent BRCA1 foci, while RAP80-IDR1-mEGFP and RAP80-(SIM+UIM)-mEGFP groups showed delayed BRCA1 recruitment. In rectal cancer tissues, positive staining of the RAP80 protein was mainly observed in the nucleus of cancer cells and high RAP80 expression was correlated with a shorter overall survival time. RAP80 undergoes LLPS to form liquid-like condensates at DSB sites, which is important for BRCA1 recruitment and enhances tumor radio-resistance.

Nuclear proinflammatory cytokine S100A9 enhances expression of human papillomavirus oncogenes via transcription factor TEAD1.

Transcription of the human papillomavirus (HPV) oncogenes, E6 and E7, is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed in vitro DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes E6 and E7 play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis.

MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop.

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H2O2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.

Development and characterization of AgHNTs@SPU film loaded with letrozole as drug delivery system and its anticancer activity

Polyurethane is an excellent implant biomaterial due to its elasticity, flexibility, resistance to abrasions and chemicals, and highly hydrophobic nature. The risk of hydrophobic nature is a significant drawback for an ideal drug delivery system. To enhance the hydrophilicity and compatibility of polyurethane via surface modification, such as (1) Introducing salicylic acid into the polymer backbone can create voids for the release of drugs from the matrix. (2) Doping silver onto HNTs by a simple one-pot precipitation method can enable silver to rupture the nucleus of cancer cells and induce ROS formation. The polyurethane and its mixed matrix were cast into the film to improve the initial effect of the drug, duration, and dosing than other conventional methods. The benefits of surface modification and thin film technology resulted in degradability, cytocompatibility, and excellent anticancer activity. The in vitro release studies depict that the letrozole-loaded film has a sustained release effect for a longer period based on the swelling ratio. The MTT assay showed that the [email protected] film could cause THP-1 cell death due to silver, polyols of PU, and HNTs. Salicylic acid played a vital role in increasing the antibacterial activity of the film. Furthermore, the increased oxidative stress was seen in [email protected]/Let film due to the biomaterial's active presence. Thus, [email protected]/Let a film is a potential approach for producing an implantable anticancer drug delivery system for the future with higher tumor cell death and prolonged drug release.

Significance of Lipopolysaccharides in Gastric Cancer and Their Potential as a Biomarker for Nivolumab Sensitivity.

Lipopolysaccharides are a type of polysaccharide mainly present in the bacterial outer membrane of Gram-negative bacteria. Recent studies have revealed that lipopolysaccharides contribute to the immune response of the host by functioning as a cancer antigen. We retrospectively recruited 198 patients with gastric cancer who underwent surgery. The presence of lipopolysaccharides was determined using immunohistochemical staining, with the intensity score indicating positivity. The relationship between lipopolysaccharides and CD8, PD-L1, TGFBI (a representative downstream gene of TGF-β signaling), wnt3a, and E-cadherin (epithelial-mesenchymal transition marker) was also investigated. Thereafter, we identified 20 patients with advanced gastric cancer receiving nivolumab and investigated the relationship between lipopolysaccharides and nivolumab sensitivity. After staining for lipopolysaccharides in the nucleus of cancer cells, 150 negative (75.8%) and 48 positive cases (24.2%) were found. The lipopolysaccharide-positive group showed increased cancer stromal TGFBI expression (p < 0.0001) and PD-L1 expression in cancer cells (p = 0.0029). Lipopolysaccharide positivity was significantly correlated with increased wnt3a signaling (p = 0.0028) and decreased E-cadherin expression (p = 0.0055); however, no significant correlation was found between lipopolysaccharide expression and overall survival rate (p = 0.71). In contrast, high TGFBI expression in the presence of LPS was associated with a worse prognosis than that in the absence of LPS (p = 0.049). Among cases receiving nivolumab, the lipopolysaccharide-negative and -positive groups had disease control rates of 66.7% and 11.8%, respectively (p = 0.088). Lipopolysaccharide positivity was associated with wnt3a, TGF-β signaling, and epithelial-mesenchymal transition and was considered to tend to promote therapeutic resistance to nivolumab.

Optimized detection and segmentation of nuclei in gastric cancer images using stain normalization and blurred artifact removal

Histological analysis with microscopy is the gold standard to diagnose and stage cancer, where slides or whole slide images are analyzed for cell morphological and spatial features by pathologists. The nuclei of cancerous cells are characterized by nonuniform chromatin distribution, irregular shapes, and varying size. As nucleus area and shape alone carry prognostic value, detection and segmentation of nuclei are among the most important steps in disease grading. However, evaluation of nuclei is a laborious, time-consuming, and subjective process with large variation among pathologists. Recent advances in digital pathology have allowed significant applications in nuclei detection, segmentation, and classification, but automated image analysis is greatly affected by staining factors, scanner variability, and imaging artifacts, requiring robust image preprocessing, normalization, and segmentation methods for clinically satisfactory results. In this paper, we aimed to evaluate and compare the digital image analysis techniques used in clinical pathology and research in the setting of gastric cancer. A literature review was conducted to evaluate potential methods of improving nuclei detection. Digitized images of 35 patients from a retrospective cohort of gastric adenocarcinoma at Oulu University Hospital in 1987–2016 were annotated for nuclei (n = 9085) by expert pathologists and 14 images of different cancer types from public TCGA dataset with annotated nuclei (n = 7000) were used as a comparison to evaluate applicability in other cancer types. The detection and segmentation accuracy with the selected color normalization and stain separation techniques were compared between the methods. The extracted information can be supplemented by patient’s medical data and fed to the existing statistical clinical tools or subjected to subsequent AI-assisted classification and prediction models. The performance of each method is evaluated by several metrics against the annotations done by expert pathologists. The F1-measure of 0.854 ± 0.068 is achieved with color normalization for the gastric cancer dataset, and 0.907 ± 0.044 with color deconvolution for the public dataset, showing comparable results to the earlier state-of-the-art works. The developed techniques serve as a basis for further research on application and interpretability of AI-assisted tools for gastric cancer diagnosis.

Design, synthesis and anticancer evaluation of novel oncolytic peptide-chlorambucil conjugates

Nitrogen mustards (NMs) are an important class of chemotherapeutic drugs and have been widely employed for the treatment of various cancers. However, due to the high reactivity of nitrogen mustard, most NMs react with proteins and phospholipids within the cell membrane. Therefore, only a very small fraction of NMs can reach the reach nucleus, alkylating and cross-linking DNA. To efficiently penetrate the cell membrane barrier, the hybridization of NMs with a membranolytic agent may be an effective strategy. Herein, the chlorambucil (CLB, a kind of NM) hybrids were first designed by conjugation with membranolytic peptide LTX-315. However, although LTX-315 could help large amounts of CLB penetrate the cytomembrane and enter the cytoplasm, CLB still did not readily reach the nucleus. Our previous work demonstrated that the hybrid peptide NTP-385 obtained by covalent conjugation of rhodamine B with LTX-315 could accumulate in the nucleus. Hence, the NTP-385-CLB conjugate, named FXY-3, was then designed and systematically evaluated both in vitro and in vivo. FXY-3 displayed prominent localization in the cancer cell nucleus and induced severe DNA double-strand breaks (DSBs) to trigger cell apoptosis. Especially, compared with CLB and LTX-315, FXY-3 exhibited significantly increased in vitro cytotoxicity against a panel of cancer cell lines. Moreover, FXY-3 showed superior in vivo anticancer efficiency in the mouse cancer model. Collectively, this study established an effective strategy to increase the anticancer activity and the nuclear accumulation of NMs, which will provide a valuable reference for future nucleus-targeting modification of nitrogen mustards.

Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta-Actin in Liver and Breast Cancer Cells.

Bee venom is a natural mixture and candidate anti-cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β-actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β-actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA-MB-231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and β-actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of β-actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of β-actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β-actin.

Influence of chromatin remodeling molecule ARID1A on metastatic heterogeneity in triple negative breast cancer: Binding of YAP.

1085 Background: Heterogeneity is the prominent character of TNBC and one predominant cause of treatment failure. As a key nuclear SWI/SNF protein complex subunit, ARID1A plays a critical role in tumor identity determining by manipulating gene expression. The present study aimed to investigate whether and how ARID1A determined the metastatic heterogeneity of triple-negative breast cancer. Methods: This study retrospectively collected and analysed clinical and pathological data from 258 patients diagnosed with triple-negative breast cancer at Fudan University Cancer Hospital from August 2015 to June 2016 to define the prognostic ability of ARID1A.CRISPR/Cas9 or plasmids-mediated ARID1A were used to determine the ARID1A function in regulating the metastatic character in TNBC. Cell morphology, tumor invasion, and metastasis were analyzed in terms of ARID1A status both in vitro and in vivo experiments. The molecular mechanism of ARID1A determining metastasis potential of TNBC was explored by RNA-Seq and nuclear positioning. Results: ARID1A low expression was found to have independent prognostic value for poor OS (43.9 vs 53.6 months, P=0.0001) and RFS (31.0 vs 45.5 months, P=0.0029) in TNBC patients. Both nuclear and cytoplasmic protein analysis and Immunofluorescent localization assay confirm that ARID1A recruits the Hippo pathway effector YAP into nucleus in human triple negative breast cancer cells. Next we designed the YAP truncator plasmid and confirmed by Co-Immunoprecipitation that ARID1A could competitively bind to the WW domain of YAP to form the ARID1A/YAP complex. Furthermore, down-regulation of ARID1A promoted migration and invasion both in human triple negative breast cancer cells and xenograft model through Hippo/YAP signaling axis. Conclusions: Our study provides new insights into the previously unrecognized role of ARID1A in TNBC metastasis, which might provide new therapeutic options for patients with TNBC.

The association of progesterone receptor and abnormal p53 expression in endometrioid endometrial cancer.

e17611 Background: Based on the TCGA endometrial cancer study results, a clinically applicable classification that identifies distinct subgroups with prognostic signature, the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), has been built and validated. It shows that those with abnormal expression of p53 (p53abn) are the group with the worst prognosis. We attempted to explore the association of p53abn and progesterone receptor (PR) in endometrioid endometrial cancer. Methods: We included 397 consecutive endometrial cancer cases of endometrioid histology with adequate tumor tissue in the formalin-fixed, paraffin-embedded (FFPE) block and available follow-up information. This study was granted by Chang Gung Medical Foundation IRB 201702242B0D001. Sanger sequencing to assess the POLE exonuclease domain hotspot mutations of exon 9-14 and immunohistochemical staining on FFPE tumor tissue sections for p53, ER, PR, and mismatch repair proteins were performed. Immunohistochemical p53 expression of cancer cell nucleus was recorded as completely negative (0%), weakly positive (1-59%), and diffusely strong positive (&gt;60%). Tumors with weak positive p53 staining were defined as p53 wild type (p53wt), while the other two were defined as p53 abnormal (p53abn). PR scores were calculated by multiplying the average staining intensity by the tumor cell nucleus stained percentage. We arbitrarily defined a progesterone receptor score of 100 or higher as positive (PR+). Progression-free and endometrial cancer-specific overall survivals starting at the date of diagnosis were evaluated using Kaplan-Meier method and compared by log-rank test between groups. Results: PR was positive in 82% of all studied cases. Of the 48 cases with p53abn tumor, 35(73%) were PR (+). With a median follow-up of 74 months, the progression-free survival and cancer-specific survival of patients with PR+ tumors (N = 310) were 93% and 96.8%, respectively, compared with 75% and 85.7% of those with PR- tumors (N = 70) (both p &lt; 0.001, log-rank test). Among those with P53abn, PR expression was associated with a favorable progression-free survival ( p = 0.081) and better cancer-specific survival ( p = 0.044). POLEmut, POLE mutated; dMMR, deficient mismatch repair PR+, progesterone receptor score ≥100, ER+, estrogen receptor score ≥100; p53abn, abnormal p53 expression. MMR was undetermined in 32 cases, PR in 17 cases, ER in 17 cases, and p53 was undetermined in 10 cases. POLE status was detected in all cases. Cases with POLE wild type, proficient mismatch repair, progesterone score &lt; 100, estrogen score &lt; 100 and wild type p53 were not listed in this table. Conclusions: Our study showed that incorporating PR into prognostic molecular markers for endometrioid endometrial cancer might provide further risk stratification. [Table: see text]

Exploring a Nuclear-Selective Radioisotope Delivery System for Efficient Targeted Alpha Therapy.

Targeted alpha therapy (TAT) has garnered significant interest as an innovative cancer therapy. Owing to their high energy and short range, achieving selective α-particle accumulation in target tumor cells is crucial for obtaining high potency without adverse effects. To meet this demand, we fabricated an innovative radiolabeled antibody, specifically designed to selectively deliver 211At (α-particle emitter) to the nuclei of cancer cells. The developed 211At-labeled antibody exhibited a superior effect compared to its conventional counterparts. This study paves the way for organelle-selective drug delivery.

Improved Analysis of Prostate Cancer: VIM3, ATG7 and P53 Form a Complex and Activate miRNA 371a-3p.

It is not possible to differentiate prostate carcinomas sufficiently to ensure that every patient receives the right therapy. New molecular markers are needed. Our objective was to identify a complex consisting of vimentin variant 3 (VIM3), autophagy-related protein 7 (ATG7) and tumor protein p53 (TP53) in prostate cancer cells and its effect on microRNA (miR)-371a-3p. Prostate cancer cell lines (PC3, DU145, LNCaP) and the benign prostatic hyperplasia cell line BPH-1 were cultured in growth medium for 24 h, then stimulated with endothelin 1 (EDN1) (50 nM) and withaferin A (2 nM) for 24 h. Cell extracts were then analyzed by western blot. The localization of VIM3, ATG7 and TP53 in the nucleus was demonstrated with immunofluorescence staining and complex formation was demonstrated by immunoprecipitation. Cancer cell migration was analyzed with a scratch assay and agarose drop analysis. The binding of the complex to the promoter of pri-miR-371a-3p was analyzed with a non-radioactive electrophoretic mobility shift assay. VIM3 knockdown using small interfering RNA and quantitative real-time polymerase chain reaction for miR-371a-3p were performed. The complex was present in the nucleus of prostate cancer cells and in the BPH-1 cell line. EDN1 increased the levels of the complex partners and cell migration, whereas withaferin A reduced the levels of the complex partners and migration. The complex bound to the promoter of pri-miR-371a-3p and might be involved in its transcription. Transfection with miR-371a-3p increased migration of prostate cancer cells. VIM3 knockdown reduced miR-371a-3p expression. The VIM3-ATG7-P53 complex, with its stimulatory effect on miR-371a-3p, may have the potential to be a marker for improved differentiation between prostate carcinomas, allowing tailored therapy.

Active U11 Peptide Luminescent Gold Nanoclusters for Pancreatic Tumor Cell Targeting

The imaging of different intercellular regions is attracting growing interest in the fields of biosensing, drug delivery, and gene therapy for cancer treatment. We present the synthesis of luminescent gold nanoclusters (AuNCs), grafted with a PEGylated U11 peptide derivative, for tumor recognition and their capacity to label the nucleus of pancreatic cancer cells. The short peptide named C3E6U11, used to synthesize and functionalize gold nanoclusters, is composed of a tricysteine sequence with side-chain thiol residues as peptide anchors for the Au nanoclusters together with the recognition peptide domain (U11). The active targeting red- or blue-emitting AuNCs can label pancreatic cancer cells and localize preferentially in the nucleus. The presence of U11 at their surface facilitates their penetration into pancreatic cell nuclei. Finally, in vivo imaging experiments in early embryos and free-swimming juvenile zebrafish are also performed to evaluate their biodistribution.

Relationship between the Reduced Expression of Zinc Finger Protein 668 in Bladder Cancer and Its Invasiveness.

The zinc finger protein 668 (ZNF668) gene encodes a Kruppel C2H2-type zinc-finger protein with 16 C2H2-type zinc fingers. The ZNF668 gene functions as a tumor suppressor gene in breast cancer. We histologically analyzed ZNF668 protein expression in bladder cancer and examined mutations of the ZNF668 gene in 68 cases of bladder cancer. In bladder cancer, the ZNF668 protein was expressed in the nuclei of cancer cells. In bladder cancer with submucosal and muscular infiltration, the expression of ZNF668 protein was significantly lower than that without submucosal and muscular infiltration. Eight heterozygous somatic mutations were detected in exon3 in five cases, and five of the mutations resulted in amino acid sequence mutations. Mutations resulting in amino acid sequence alterations also resulted in lower ZNF668 protein expression in bladder cancer cell nuclei, but no significant association with bladder cancer infiltration was detected. Decreased ZNF668 expression in bladder cancer was associated with submucosal and muscle invasion of cancer cells. Somatic mutations resulting in amino acid mutations in ZNF668 were found in 7.3% of the bladder cancer cases.