S Phase Research Articles

Extensive DNA Damage and Loss of Cell Viability Occur Synergistically With the Combination of Recombinant Methioninase and Paclitaxel on Pancreatic Cancer Cells which Report DNA-Damage Response in Real Time.

Methionine restriction selectively arrests cancer cells during the S-phase of the cell cycle. We hypothesized that DNA damage may occur in S-phase in cancer cells during methionine restriction. To determine if this occurs, we used MiaPaCa-2Tet-On 53BP1-green fluorescent protein (GFP) pancreatic cancer cells, which report GFP fluorescence in real time after DNA-damage response (DDR) in these cells. We also determined whether a chemotherapy drug in combination with methionine restriction increases the rate of DNA damage. MiaPaCa-2Tet-On 53BP1-GFP cells were used for in vitro experiments. The 25% and 50% inhibitory concentrations (IC25 and IC50, respectively) of recombinant methioninase (rMETase) and paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP pancreatic cancer cells were determined. Cell viability and DDR with rMETase alone, paclitaxel alone, and their combination were measured in MiaPaCa-2Tet-On 53BP1-GFP cells. The IC25 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 1.66 U/ml. The IC25 for paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP cells was 3.31 nM. The combination of rMETase and paclitaxel synergistically reduced the viability of MiaPaCa-2Tet-On 53BP1-GFP cells. The IC50 of paclitacel on MiaPaCa-2Tet-On 53BP1-GFP cells was 5.1 nM. The IC50 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 2.3 U/ml. The combination of rMETase (IC50) plus paclitaxel (IC50) on MiaPaCa-2Tet-On 53BP1-GFP cells also caused more DNA damage than either agent alone. The present study suggests the synergy of methionine restriction and chemotherapy is due, at least in part, to DNA damage of cancer cells.

QSOX1 Modulates Glioblastoma Cell Proliferation and Migration In Vitro and Invasion In Vivo.

Background: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme that catalyzes the oxidation of free thiols to generate disulfide bonds in a variety of proteins, including the cell surface and extracellular matrix. QSOX1 has been reported to be upregulated in a number of cancers, and the overexpression of QSOX1 has been correlated with aggressive cancers and poor patient prognosis. Glioblastoma (GBM) brain cancer has been practically impossible to treat effectively, with cells that rapidly invade normal brain tissue and escape surgery and other treatment. Thus, there is a crucial need to understand the multiple mechanisms that facilitate GBM cell invasion and to determine if QSOX1 is involved. Methods and Results: Here, we investigated the function of QSOX1 in human glioblastoma cells using two cell lines derived from T98G cells, whose proliferation, motility, and invasiveness has been shown by us to be dependent on disulfide bond-containing adhesion and receptor proteins, such as L1CAM and the FGFR. We lentivirally introduced shRNA to attenuate the QSOX1 protein expression in one cell line, and a Western blot analysis confirmed the decreased QSOX1 expression. A DNA content/cell cycle analysis using flow cytometry revealed 27% fewer knockdown cells in the S-phase of the cell cycle, indicating a reduced proliferation. A cell motility analysis utilizing our highly quantitative SuperScratch time-lapse microscopy assay revealed that knockdown cells migrated more slowly, with a 45% decrease in migration velocity. Motility was partly rescued by the co-culture of knockdown cells with control cells, indicating a paracrine effect. Surprisingly, knockdown cells exhibited increased motility when assayed using a Transwell migration assay. Our novel chick embryo orthotopic xenograft model was used to assess the in vivo invasiveness of knockdown vs. control cells, and tumors developed from both cell types. However, fewer invasive knockdown cells were observed after about a week. Conclusions: Our results indicate that an experimental reduction in QSOX1 expression in GBM cells leads to decreased cell proliferation, altered in vitro migration, and decreased in vivo invasion.

Gemcitabine: Association with Thrombocytosis

Gemcitabine is a pyrimidine antimetabolite that affects DNA synthesis in the S-phase of the cell cycle. It is effective against cancers such as ovarian carcinoma, non-small cell lung cancer, and pancreatic cancer. However it causes significant side effects like myelosuppression, which leads to thrombocytopenia in about 71% of patients, prompting discontinuation of therapy. Even though thrombocytopenia is common, some patients experience thrombocytosis during Gemcitabine therapy, especially in the second and third cycles, possibly due to a rebound effect. Thrombocytosis is usually asymptomatic and occasionally leads to venous thrombosis, but its clinical significance is uncertain because of conflicting studies. Further research is required to understand the physiology behind thrombocytosis in Gemcitabine-treated patients. Fluctuations between thrombocytosis and thrombocytopenia need to be closely monitored. Management for Gemcitabine-induced thrombocytosis can be improved if more comprehensive studies are undertaken to explore the role of thrombocytosis in cardiovascular events.

Aurora A Kinase Begins to Localize to the Centrosome in the S-phase of the Cell Cycle in the XL2 Cell Line

Background: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance. Methods: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work. Results: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells. Conclusions: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.

Abstract CT233: Serum thymidine kinase 1 levels as a predictive marker for castration resistance in metastatic prostate cancer

Abstract Introduction Thymidine kinase 1 (TK1) is an enzyme associated with the cell division cycle; its levels increase during the S-phase of the cell cycle and decrease during the M-phase. In cancer TK1 release is increased both by increased cell proliferation and cell death. Thus, serum concentration of TK1 reflects the tumor volume, proliferation rate, and extent of cell death. Serum Thymidine Kinase 1 (sTK1) enzyme has been used as a prognostic, predictive, and monitoring biomarker in leukemia, Hodgkin's and non-Hodgkin's lymphomas. Additionally, sTK1 enzyme levels have been elevated in breast cancer and prostate cancer. This study evaluated whether sTK1 predicts the risk of castration resistance in patients with metastatic prostate cancer undergoing androgen deprivation therapy (ADT). Materials and Methods sTK1 levels was measured in men diagnosed with metastatic prostate cancer at 3 time points; before the initiation of ADT, 6 months after the start of treatment, and within 6 months before the development of castration resistance. Samples at study visits of ESTO2 trial, which compares atorvastatin to placebo in combination with ADT. The trial intervention was blinded at analysis. Cox regression modeling was used to assess whether sTK1 values predict the risk of castration resistance. sTK1 was analyzed both as continuous and categorical variable, stratified by median value 0.12 ng/ml. The time from the initiation of ADT to the formation of castration resistance, death, or a general index date (May 31, 2023) was used as the time-metric. Results The study included serum samples from 70 patients, of which 23 had developed castration resistance by May 31,2023. The median follow-up time was 13 months. The sTK1 value measured before ADT initiation did not predict the risk of castration resistance (HR 0.91, 95% CI 0.62-1.35). However, sTK1 at 6 months after the initiation of ADT or up to a maximum of 6 months before formation of castration resistance did predict the risk (HR 1.5, 95% CI 1.07-2.1 and HR 1.79, 95% CI 1.16-2.76, respectively). sTK1 predicted the risk at both measurement points also when analyzed as categorical variable. sTK1 was predictive only among men with metastatic disease; sTK1 no longer predicted the risk of castration resistance who started ADT for biochemically recurrent, non-metastatic disease. Conclusions Our findings suggest that sTK1 value can be used to predict the risk of progression to castration resistance during ADT in metastatic prostate cancer. However, these findings need validation from larger datasets. Citation Format: Aino Siltari, Paavo Raittinen, Jarno Riikonen, Juha Koskimäki, Tomi Pakarainen, Mikael Anttinen, Otto Ettala, Teuvo Tammela, Stig Linder, Teemu J. Murtola. Serum thymidine kinase 1 levels as a predictive marker for castration resistance in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT233.

Abstract 5785: Shedding light on PARP1-E2F1 interaction by Cryo-EM

Abstract Cell division is orchestrated by a complex network of interactions between proteins involved in numerous signaling pathways that control cell proliferation. One of these main players is the E2F family of transcription factors. The role of these key players is to regulate the expression of several genes important in cell proliferation, particularly those involved in progression through G1 and into the S-phase of the cell cycle. In fact, cell cycle deregulation is a hallmark of cancer, and multiple studies have highlighted the critical role of E2F hyperactivation in tumor progression, angiogenesis, and metastasis. However, targeting a master regulator of cell growth will also have a pernicious effect on non-cancerous cells, thus limiting its applications. In order to overwhelm these restrictions, we propose a therapeutic approach in which E2F transcriptional activity is specifically reduced in cells with hyperactivation of the pathway, such as cancer cells.We and others have shown that poly (ADP-ribose) polymerase 1 (PARP1) is a transcriptional co-activator of E2F1 and, owing to this, loss of PARP results in cell cycle re-regulation and reduction of tumor growth. Interestingly, this effect of PARP1 does not depend on its enzymatic activity, thus opening a completely new strategic approach. Furthermore, considering that most oncogenic processes are associated with cell cycle deregulation, disruption of PARP1-E2F1 interaction could provide a new therapeutic target with a wide spectrum of indications in cancer. Currently, we lack comprehensive structural details regarding the PARP-1/E2F1 protein-protein interaction. To address this, we used cryo-EM to investigate both the standalone structure of PARP1 (full length) and its interaction with E2F1. This approach yielded high-resolution structural insights into the PARP1-E2F1 complex, enabling precise identification of the protein-protein interaction interface (PPI). Through this exploration of structural and molecular determinants governing this interaction, we aim to develop innovative strategies for designing drugs targeted at disrupting this complex. This work was supported by Agencia Estatal de Investigación (AEl/10.13039/501100011033), Xunta de Galicia (GPC GI-1862, ED431B 2020/26; ED431G 2019/02) and European Regional Development Fund-ERDF. Citation Format: Pablo Iglesias, Ester Casajus-Pelegay, Maria Moreno-Morcillo, Lara González-Rendo, Laura Porres-Ventin, Victor M. Arce, Rafael Fernandez-Leiro, Jose A. Costoya. Shedding light on PARP1-E2F1 interaction by Cryo-EM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5785.

DIVERSITY OF H2A HISTONES AND THEIR IMPLICATIONS FOR NUCLEOSOME STRUCTURAL PROPERTIES

Histone proteins are key epigenetic factors, which play an important role in chromatin dynamics and gene activity regulation. They are divided into two broad classes: canonical histones and their variants. Canonical histones are expressed mainly during the S-phase of the cell cycle, as they are involved in DNA packaging during cell division. Histone variants are histone genes that are expressed and regulate chromatin dynamics throughout the cell cycle. Due to the functional and species diversity, various families of histone variants are distinguished. Some proteins may diff er slightly from canonical histones, while others, on the contrary, may have many important structural and functional features that aff ect nucleosome stability and chromatin dynamics. In order to assess the variability of the H2A histone family and their role in nucleosome structure, we performed a bioinformatic analysis of the amino acid sequences of the H2A histone family. The clustering performed by the UPGMA method made it possible to reveal two main subfamilies of H2A proteins: short H2A and other H2A variants demonstrating highly conserved amino acid sequences. We also constructed and analyzed multiple alignments for various H2A histone subfamilies. It is important to note that the proteins of the short H2A subfamily are not only the least conserved within the H2A family, but also have features that signifi cantly aff ect the structural properties of the nucleosome. In addition, we performed a phylogenetic analysis of short H2A, which resulted in the identifi cation and characterization of individual clades on the phylogenetic tree for the variants H2A.B, H2A.P, H2A.Q, H2A.L.

Abstract B027: Repurposing ion channel drugs: the Na+ channel modifying drug DPI-201-106 significantly enhances the anti-tumor activity of multiple classes of drugs targeting DNA damage repair

Abstract B027: Repurposing ion channel drugs: the Na+ channel modifying drug DPI-201-106 significantly enhances the anti-tumor activity of multiple classes of drugs targeting DNA damage repair

CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone mRNA processing.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited effective treatment options, potentiating the importance of uncovering novel drug targets. Here, we target cleavage and polyadenylation specificity factor 3 (CPSF3), the 3' endonuclease that catalyzes mRNA cleavage during polyadenylation and histone mRNA processing. We find that CPSF3 is highly expressed in PDAC and is associated with poor prognosis. CPSF3 knockdown blocks PDAC cell proliferation and colony formation in vitro and tumor growth in vivo. Chemical inhibition of CPSF3 by the small molecule JTE-607 also attenuates PDAC cell proliferation and colony formation, while it has no effect on cell proliferation of nontransformed immortalized control pancreatic cells. Mechanistically, JTE-607 induces transcriptional readthrough in replication-dependent histones, reduces core histone expression, destabilizes chromatin structure, and arrests cells in the S-phase of the cell cycle. Therefore, CPSF3 represents a potential therapeutic target for the treatment of PDAC.

2-methoxyestradiol inhibits the malignant behavior of triple negative breast cancer cells by altering their miRNome.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with no effective targeted treatment currently available. Estrogen and its metabolites influence the growth of mammary cancer. Previously, we demonstrated the anti-cancer effects of 2-methoxyestradiol (2ME2) on mammary carcinogenesis. In the present study, we investigated the effects of 2ME2 on TNBC cells. TNBC (MDA-MB-231 and MDA-MB-468) and non-tumorigenic breast (MCF10A) cell lines were used to determine the effects of 2ME2 on cell proliferation (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS assay), cell cycle (flow cytometric assay), migration (transwell migration assay), invasion (matrigel invasion assay), apoptosis (annexin V/propidium iodide assay), colony formation (soft agar assay), and miRNome (human miRNA profiling array). The miRNome data were analyzed using the c-BioPortal and Xena platforms. Moreover, Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and reactome pathway analyses were performed. We found that 2ME2 effectively inhibited cell proliferation and induced apoptosis. Furthermore, 2ME2 treatment arrested TNBC cells in the S-phase of the cell cycle. Treatment with 2ME2 also significantly decreased the aggressiveness of TNBC cells by inhibiting their migration and invasion. In addition, 2ME2 altered the miRNA expression in these cells. In silico analysis of the miRNome profile of 2ME2-treated MDA-MB-468 cells revealed that miRNAs altered the target genes involved in many different cancer hallmarks. 2ME2 inhibits triple negative breast cancer by impacting major cellular processes like proliferation, apoptosis, metastasis, etc. It further modifies gene expression by altering the miRNome of triple negative breast cancer cells. Overall, our findings suggest 2ME2 as a potent anti-cancer drug for the treatment of TNBC.

Clinicohistopathological Features and Immunohistochemical Expression Patterns of p53 in Epithelial Ovarian Tumours: A Cross-sectional Study

Introduction: Ovarian tumours represent 3% of female malignancies, with epithelial tumours constituting more than 90% of all ovarian cancers. Tumour suppressor gene 53 (TP53) mutations play an important role in the prognosis and treatment of ovarian cancer. The tumour suppressor gene Tumour Protein 53 (p53), located on the short arm of chromosome 17, acts by suppressing abnormal cell growth at the beginning of the Synthesis phase (S-phase) of the cell cycle. Previous studies have shown a correlation between p53 mutation or overexpression and patient prognosis in various types of tumours, including breast cancer, rectal cancer, intestinal cancer, lung cancer, and ovarian cancer. Aim: To investigate the clinicohistopathological features and immunohistochemical expression of p53 in Epithelial Ovarian Tumours (EOT). Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at Hind Institute of Medical Sciences, Barabanki, Uttar Pradesh, India. The duration of the study was one year and two months, from September 2021 to November 2022. A total of 80 cases of EOT were included and histopathological diagnosis was performed, and immunohistochemical expression of p53 was evaluated in all cases. The Chi-square test was used to compare categorical data (the number of benign, borderline, and malignant EOT) and determine whether the difference in p53 expression (positive or negative) was statistically significant, using Statistical Package for the Social Sciences (SPSS) version 26.0. Results: The total of 80 cases of EOT in the present study comprised 56 (70%) benign, 5 (6.25%) borderline, and 19 (23.75%) malignant tumours. The p53 expression was statistically significant. Immunohistochemistry (IHC) for p53 showed diffuse strong positive nuclear staining (>60%) of the tumour cells in 14 cases, all of which were malignant epithelial tumours. Focally weak and patchy positive nuclear staining patterns (5%-60%) were observed in 64 cases, including 56 benign cases, four borderline cases, and four cases of malignant epithelial tumours, respectively. p53 positivity (<5% staining of tumour cells) was seen in two cases, one each of borderline and malignant epithelial tumour, respectively. Conclusion: The IHC marker p53 serves as a surrogate marker for p53 gene mutation, and its positivity is observed in serous EOT. Understanding p53 staining patterns can be used in conjunction with a panel of other antibodies to correctly classify morphologically confusing EOT. This can aid in assessing prognosis and understanding the biological behaviour of the tumour, which can be helpful in modifying the treatment plan.

Modeling Retinoblastoma in adult Drosophila by reducing Rbf gene expression

Retinoblastoma is an eye cancer that begins in the retina due to genetic mutations in the retinal cells, typically of the Rb1 tumor suppressor gene, which encodes for the Rb protein. Rb inhibits the E2F transcription factor, which drives the expression of genes needed for entry into S-phase of the cell cycle. Loss of function of Rb dysregulates E2F, causing uninhibited cell-cycle proliferation and tumor formation. Current treatments for retinoblastoma are highly invasive such as surgery to remove the eye or chemotherapy impacting patients' lives. If an animal model of retinoblastoma is constructed, less invasive therapies can be tested. Drosophila melanogaster have a short life cycle, are cost-effective, and have an Rb1 homologue called Rbf1, making them an efficient model. An RNAi system was used to knock down the expression of Rbf1 protein via the gsGAL4-UAS gene switch system of gene regulation. A fly line in which the gsGAL4 transcription factor is expressed under the eye-specific elav promoter was crossed with a line in which an siRNA targeting Rbf1 mRNA is expressed under the control of an upstream activating sequence (UAS). When the offspring of this cross were fed RU486, the normally cytoplasmic gsGAL4 entered the nucleus of the retinal cells in which it was expressed and bound to the UAS, driving the expression of the siRNA, which was expected to knock down levels of Rbf1 protein in these flies. RU486-treated flies exhibited a change in eye morphology, suggestive of tumor formation, as well as differential Kaplan-Meier survival curves.

A Phase I Study of Ruxolitinib in Combination with Abemaciclib for Patients with Primary or Post-Polycythemia Vera/Essential Thrombocythemia Myelofibrosis

Background and Significance: Patients (pts) with primary or secondary myelofibrosis (MF) experience variable degrees of constitutional symptoms, cytopenias and risk of progression to acute myeloid leukemia leading to a reduced life-expectancy compared to age-matched controls. Although several oral Janus Kinase (JAK) inhibitors such as ruxolitinib (RUX) have been approved by the US FDA based on improvements in symptom burden and spleen volume compared to physician's choice of best available therapy, the duration of response to these agents is generally time-limited and treatment with JAK inhibitors is not considered to have disease-modifying potential. Furthermore, the median overall survival after RUX discontinuation is only 11-14 months highlighting the need for novel mechanism-based therapies for pts with disease progression on RUX. JAK2 V617F mutations promote transition from the G1 to S-phase of the cell cycle via increased expression of CDC25A, which is also upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been implicated in MF pathogenesis and contribute to myeloproliferation. Finally, we and others have shown synergistic effects of the combination of CDK4/6 inhibitors and RUX in murine models of MF (Rampal et al., CCR 2021) including improvement in bone marrow fibrosis suggesting the potential for disease-modifying activity. Several CDK4/6 inhibitors have been approved by the US FDA for the treatment of hormone receptor-positive metastatic breast cancer establishing a robust record of safety data. These preclinical data and known safety profile of CDK4/6 and RUX as single agents, provide rationale for combining CDK4/6 and JAK inhibition in pts with MF. Methods: This multicenter, phase I dose-escalation trial (NCT05714072) evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in primary or secondary MF pts with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. Inadequate response to RUX is defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms, as defined by the presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS. Pts must have been on a stable dose of 10 mg or 15 mg BID of RUX for at least 4 weeks prior to enrollment with adequate platelet (≥75 x 10 9/L) and neutrophil counts (≥1.5 x 10 9/L). Key exclusion criteria include prior treatment with a CDK4/6 inhibitor, accelerated- or blast-phase MPN, platelet count <50 x 10 9/L in the 4 weeks before screening or platelet transfusion(s) within 8 weeks before screening, and absolute neutrophil count <0.5 x 10 9/L in the 4 weeks before screening. Among the currently FDA-approved CDK4/6 inhibitors, abemaciclib was chosen for combination therapy due to less myelosuppressive effects compared with other CDK4/6 inhibitors. This trial uses a conventional “3+3” dose-escalation design ( Figure), pts will be treated with increasing doses of abemaciclib added to a stable dose of RUX (10 mg or 15 mg BID). The pre-study dose of RUX will be maintained to determine the maximum tolerated dose of the combination of RUX and abemaciclib. The primary endpoint is to determine the maximum tolerated dose of RUX + abemaciclib. Secondary endpoints include measures of efficacy such as overall response rate, duration of response and survival endpoints. Exploratory endpoints include changes in allele fraction of driver mutations, gene expression profiling, and clonal architecture to identify biomarkers of response, mechanisms of resistance, and to assess for any disease-modifying effects of the combination therapy. At this time, 2 pts have been enrolled at dose level 0 without any dose-limiting toxicities observed. In line with the known adverse event profile of abemaciclib, grade 1 diarrhea occurred in one patient. One patient developed grade 3 neutropenia which promptly resolved with holding abemaciclib. No unexpected safety signal was noted and enrollment continues. This trial is open to enrollment at Memorial Sloan Kettering Cancer Center and will commence at MD Anderson Cancer Center later this year.

Study on in vitro therapeutic impact of 17-ꞵ Estradiol on Glioblastoma cell lines

Glioblastomas (GBM) or grade IV astrocytomas is the most aggressive and frequent tumor in the Central Nervous System (CNS). GBM treatment mainly consists of surgical resection and radio and/or chemotherapy. However, due to its infiltration capacity, it is practically impossible to completely extract the tumor and it relapses. Aberrant EGFR expression is observed in GBM including EGFR mutation, rearrangement and deletions. Blood brain barrier is one of the major obstacles which prevents the entry of larger molecules into the brain. Hence a small molecule, with lesser neurotoxicity should be developed in treating GBM or any other brain related diseases. Previous studies have reported the therapeutic potency of 17-ꞵ Estradiol in treating breast cancer, but little is known about the therapeutic role of estrogen signalling in the development and progression of gliomas. Estradiol being the small molecule has molecular weight of 272.4g/mol. It is a lipophilic molecule and therefore it can pass through the blood brain barrier by passive diffusion and could be considered as a therapeutic molecule in treating GBM. Present study showed that 17-ꞵ Estradiol inhibited the migration, colony formation in GBM cell lines. Further it induced early and late apoptosis and arrested the cell cycle at S-Phase of the cell cycle. EGFR and downstream EGFR associated protein expression were also reduced upon exposure of GBM cell lines to E2. These results show that 17-ꞵ Estradiol could be considered as a therapeutic molecule in GBM treatment.

Initiation of Apoptotic Pathway by the Cell-Free Supernatant Synthesized from Weissella cibaria Through In-Silico and In-Vitro Methods.

Globally, colorectal cancer is the most prevalent type of cancer. Even though multiple treatments such as surgery, radiation, chemotherapy, and immunotherapy are available, the adverse effects caused in patients seem remarkable. Therefore, the current work was deliberated to prepare the metabolites (cell-free supernatant-CFS) from Weissella cibaria RK-3-1 to conduct in-silico and in-vitro-based anticancer assays. First, the active biomolecules present in the CFS were screened using a GC-MS analyzer. In addition, in-silico-based pharmacokinetic and docking studies were performed to confirm the anticancer potential of metabolites. In-silico results suggested that the bioactive compounds such as filicinic acid, dibutyl phthalate, and 4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl present in CFS possessed significant molecular docking interactions with anticancer hub proteins. Furthermore, in-vitro results displayed the inhibition of cell proliferation in HT-29 cells at an IC50 value of 22.5 ± 1.3µg/ml with the least significant effect on HEK-293 cell lines. Moreover, bacterial metabolites-controlled cell proliferation during the cell cycle's synthesis phase (S). Furthermore, the gene expression results confirm the increased expression of Bad, Bax, Bcl2, caspase-3, and cytochrome-C genes involved in the intrinsic apoptotic pathway. Hence, our findings from the in-silico and the in-vitro study confirm the anticancer potential of cell free-supernatant synthesized by W. cibaria.

The minichromosome maintenance complex drives esophageal basal zone hyperplasia.

Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen-driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography-tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell-related proteins were among the most highly upregulated DEPs in EoE compared with controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all 6 subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of epithelial cells through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.

Design, synthesis and biological evaluation of a novel bioactive indane scaffold 2-(diphenylmethylene)c-2,3-dihydro-1H-inden-1-one with potential anticancer activity

Over the past decades, designing of privileged structures has emerged as a useful approach to the discovery and optimisation of novel biologically active molecules, and many have been successfully exploited across and within different target families. Examples include indole, quinolone, isoquinoline, benzofuran and chromone, etc. In the current study, we focus on synthesising a novel hybrid scaffold constituting naturally occurring benzophenone (14) and indanone (22) ring systems, leading to a general structure of 2-(diphenylmethylene)-2,3-dihydro-1H-inden-1-one (23). It was hypothesised this new hybrid system would provide enhanced anti-cancer activity owing to the presence of the common features associated with the tubulin binding small molecule indanocine (10) and the estrogen receptor (ER) antagonist tamoxifen (24). Key hybrid molecules were successfully synthesised and characterised, and the in vitro cytotoxicity assays were performed against cancer cell lines: MCF7 (breast) and SKBR3 (breast), DU145 (prostate) and A549 (lung). The methyl-, chloro- and methoxy-, para-substituted benzophenone hybrids displayed the greatest degree of cytotoxicity and the E-configuration derivatives 45, 47 and 49 being significantly most potent. We further verified that the second benzyl moiety of this novel hybrid scaffold is fundamental to enhance the cytotoxicity, especially in the SKBR3 (HER2+) by the E-methyl lead molecule 47, MCF7 (ER+) by 45 and 49, and A549 (NSCLC) cell lines by 49. These hybrid molecules also showed a significant accumulation of SKBR3 cells at S-phase of the cell cycle after 72 hrs, which demonstrates besides of being cytotoxic in vitro against SKBR3 cells, 47 disturbs the replication and development of this type of cancer causing a dose-dependent cell cycle arrest at S-phase. Our results suggest that DNA damage might be involved in the induction of SKBR3 cell death caused by the hybrid molecules, and therefore, this novel system may be an effective suppressor of HER2+/Neu-driven cancer growth and progression. The present study points to potential structural optimisation of the series and encourages further focussed investigation of analogues of this scaffold series toward their applications in cancer chemoprevention or chemotherapy.

5-Fluorouracil-loaded designed praseodymium oxide – poly- β-cyclodextrin nanorods for effectively inhibiting breast cancer cells

Breast cancer remains one of the commonly prevailing and challenging diseases. Lanthanide-based nanomaterials are fascinating depending on their unique properties and represent a preferable choice of drug transport, aimed at finding an agent for targeted therapy. It necessitates that lanthanide-based nanocarriers with suitable surface modification and desired physicochemical characteristics are developed for effective anticancer drug delivery, owing to their fascinating light absorption, magnetic, and morphological features. Herein, we report praseodymium oxide nanorods in combination with poly-cyclodextrin as a novel nanocarrier to deliver the chemotherapeutic drug 5-fluorouracil to breast cancer cells. The praseodymium oxide nanoparticles were prepared using a sonochemical method and characterized using routine analytical methods, including X-ray Diffraction, Transmission Electron Microscopy, and X-ray Photoelectron Spectroscopy. The monodisperse rod-shaped structures possess length and width of 200 ± 30 and 72 ± 10 nm, respectively. The nanoparticles are 64.33% crystalline, revealed by XRD. The polymer content in the nanoparticles is ∼37% by weight. Further, the nanoparticles' magnetic, light absorption and photoluminescence characteristics are studied. Low saturation magnetization and insignificant hysteresis properties are observed. Broad absorption and visible wavelength region photoluminescence arise from the praseodymium in the material. The continuous release of the drug cargo and the in vitro anticancer activity are discussed. The loaded drug (5-fluorouracil) is released sustainably, extending over 250 hours. The anticancer activity in vitro is observed with the portion of the drug released from the nanocarrier. The apoptotic action mode of the drug-carrier vehicle is analyzed. The inhibition of cells occurs mainly on the S-phase of the cell cycle. The nanomaterial can function as a drug carrier and is suitable for anticancer therapy. The results reported herein on praseodymium oxide nanoparticles are promising as a nanocarrier and pose the possibility of future in vivo studies.

The Role of Low-Density Lipoprotein Receptor-Associated Protein LRP11 in the Development of Hepatocellular Carcinoma

This study investigated the role of the low-density lipoprotein receptor-associated protein LRP11 in hepatocellular carcinoma (HCC). Analysis of TCGA and GTEx databases revealed that LRP11 expression was significantly increased in liver cancer tissues compared to normal tissues (P <0.05). High expression of LRP11 was associated with shorter survival in liver cancer patients (P <0.05). Lentivirus transfection was used to create sh-NC and sh-LRP11 groups for further experiments. Silencing LRP11 in HepG2 cells resulted in a significant decrease in cell viability (P <0.05), increased apoptosis rate (P <0.01), and upregulation of the apoptosis-related protein Bax (P <0.01) and downregulation of Bcl-2 (P <0.01). Moreover, the sh-LRP11 group showed a significant decrease in the S-phase of the cell cycle (P <0.01) and reduced expression of Cyclin D1 (P <0.01). These findings indicate that LRP11 is highly expressed in liver cancer tissues and is associated with an unfavorable prognosis. Suppression of LRP11 expression inhibits the proliferation of HCC cells, promotes apoptosis, and affects cell cycle progression. These results contribute to understanding the molecular mechanisms underlying HCC development and progression, as well as identifying potential therapeutic targets.

Methods for Inferring Cell Cycle Parameters Using Thymidine Analogues.

Tritiated thymidine autoradiography, 5-bromo-2'-deoxyuridine (BrdU) 5-chloro-2'-deoxyuridine (CldU), 5-iodo-2'-deoxyuridine (IdU), and 5-ethynyl-2'-deoxyiridine (EdU) labeling have been used for identifying the fraction of cells undergoing the S-phase of the cell cycle and to follow the fate of these cells during the embryonic, perinatal, and adult life in several species of vertebrate. In this current review, I will discuss the dosage and times of exposition to the aforementioned thymidine analogues to label most of the cells undergoing the S-phase of the cell cycle. I will also show how to infer, in an asynchronous cell population, the duration of the G1, S, and G2 phases, as well as the growth fraction and the span of the whole cell cycle on the base of some labeling schemes involving a single administration, continuous nucleotide analogue delivery, and double labeling with two thymidine analogues. In this context, the choice of the optimal dose of BrdU, CldU, IdU, and EdU to label S-phase cells is a pivotal aspect to produce neither cytotoxic effects nor alter cell cycle progression. I hope that the information presented in this review can be of use as a reference for researchers involved in the genesis of tissues and organs.