Cell Cycle Phase Distribution Research Articles

PLK1 Inhibitor Onvansertib Enhances the Efficacy of Alpelisib in PIK3CA-Mutated HR-Positive Breast Cancer Resistant to Palbociclib and Endocrine Therapy: Preclinical Insights.

Endocrine therapy (ET) combined with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) is the preferred first-line treatment for hormone receptor-positive (HR+)/HER2- metastatic breast cancer. Although this is beneficial, acquired resistance leads to disease progression, and patients harboring PIK3CA mutations are treated with targeted therapies such as the PI3Kα inhibitor, alpelisib, alongside ET. Drug-associated resistance mechanisms limit the efficacy of alpelisib, highlighting the need for better combination therapies. This study aimed to evaluate the efficacy of combining alpelisib with a highly specific PLK1 inhibitor, onvansertib, in PIK3CA-mutant HR+ breast cancer preclinical models. We assessed the effect of the alpelisib and onvansertib combination on cell viability, PI3K signaling pathway, cell cycle phase distribution and apoptosis in PI3K-activated HR+ breast cancer cell lines. The antitumor activity of the combination was evaluated in three PIK3CA-mutant HR+ breast cancer patient-derived xenograft (PDX) models, resistant to ET and CDK4/6 inhibitor palbociclib. Pharmacodynamics studies were performed using immunohistochemistry and Simple Western analyses in tumor tissues. The combination synergistically inhibited cell viability, suppressed PI3K signaling, induced G2/M arrest and apoptosis in PI3K-activated cell lines. In the three PDX models, the combination demonstrated superior anti-tumor activity compared to the single agents. Pharmacodynamic studies confirmed the inhibition of both PLK1 and PI3K activity and pronounced apoptosis in the combination-treated tumors. Our findings support that targeting PLK1 and PI3Kα with onvansertib and alpelisib, respectively, may be a promising strategy for patients with PIK3CA-mutant HR+ breast cancer failing ET + CDK4/6i therapies and warrant clinical evaluation.

Perturbed cell cycle phase-dependent positioning and nuclear migration of retinal progenitors along the apico-basal axis underlie global retinal disorganization in the LCA8-like mouse model

Combined removal of Crb1 and Crb2 from the developing optic vesicle evokes cellular and laminar disorganization by disrupting the apical cell-cell adhesion in developing retinal epithelium. As a result, at postnatal stages, affected mouse retinas show temporarily thickened, coarsely laminated retinas in addition to functional deficits, including a severely abnormal electroretinogram and decreased visual acuity. These features are reminiscent of Leber congenital amaurosis 8, which is caused in humans by subsets of Crb1 mutations. However, the cellular basis of the abnormalities in retinal progenitor cells (RPCs) that lead to retinal disorganization is largely unknown. In this study, we analyze specific features of RPCs in mutant retinas, including maintenance of the progenitor pool, cell cycle progression, cell cycle phase-dependent nuclear positioning, cell survival, and generation of mature retinal cell types. We find crucial defects in the mutant RPCs. Upon removal of CRB1 and CRB2, apical structures of the RPCs, determined by markers of cilia and centrosomes, are basally shifted. In addition, the positioning of the somata of the M-phase cells, normally localized at the apical surface of the retinal epithelium, is basally shifted in a nearly randomized pattern along the apico-basal axis. Consequently, we propose that positioning of RPCs is desynchronized from cell cycle phase and largely randomized during embryonic development at E17.5. Because the resultant postmitotic cells inevitably lose positional information, the outer and inner nuclear layers (ONL and INL) fail to form from ONBL during neonatal development and retinal cells become mixed locally and globally. Additional results of the lost tissue polarity in Crb1/Crb2 dKO retinas include atypical formation of heterotopic cell patches containing photoreceptor cells in the ganglion cell layer and acellular patches filled with neural processes. Collectively, these changes lead to a mouse model of LCA8-like pathology. LCA8-like pathology differs substantially from the well-characterized, broad range of degeneration phenotypes that arise during the differentiation of photoreceptor and Muller glial cells in retinitis pigmentosa 12, a closely related disease caused by mutated human Crb1.Importantly, the present results suggest that Crb1/Crb2 serve indispensable functions in maintaining cell-cycle phase-dependent positioning of RPCs along the apico-basal axis, regulating cell cycle progression, and maintaining structural laminar integrity without significantly affecting the size of the RPC pools, generation of the subsets of the retinal cell types, or the distribution of cell cycle phases during RPC division. Taken together, these findings provide the crucial cellular basis of the thickening and severely disorganized lamination that are the unique features of the retinal abnormalities in LCA8 patients.

Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib)

BackgroundBRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors.MethodsCell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array.ResultsThe resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRβ receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion.ConclusionsIn summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.

Synthesis, in vitro anticancer activity, and pharmacokinetic profiling of the new tetrahydropyrimidines:Part I.

Different vanillin-based aldehydes were used to synthesize novel tetrahydropyrimidines (THPMs) via conventional Biginelli reaction. The THPMs were tested against human normal cells (MRC-5) and cancer cell lines (HeLa, K562, and MDA-MB-231). With IC50 values of 10.65, 10.70, and 12.76 µM, compounds 4g, 4h, and 4i exerted the strongest cytotoxic effects against K562 cells. The best activity was achieved for 4g on MDA-MB-231 cells (IC50 = 9.20 ± 0.14 µM). The effects of compounds 4g, 4h, and 4i on the cell-cycle phase distribution of K562 cells were analyzed. Principal component analysis was carried out for the chemometrics analysis to comprehend the relationship between the anticancer activity of the THPMs, pharmacokinetic properties, and partition coefficients, as well as the relationship between the chromatographic behavior and retention parameters. The highest retention rates are found for molecules 4g, 4h, and 4i, which have the longest carbon chains, indicating that the length of the alkyl chain positively affects the molecule's anticancer activity but only if the number of carbon atoms is not higher than seven. Additionally, molecular docking analysis was performed to determine the preferred binding modes of the investigated ligands (4g, 4h, and 4i) with a DNA dodecamer and bovine serum albumin.

Light-activated CRISPR-Cas12a for amplified imaging of microRNA in cell cycle phases at single-cell levels.

An ortho-nitrobenzyl phosphate ester-caged nucleic acid hairpin structure coupled to the CRISPR-Cas12a complex is introduced as a functional reaction module for the light-induced activation of the CRISPR-Cas12a (LAC12a) machinery toward the amplified fluorescence detection of microRNA-21 (miRNA-21). The LAC12a machinery is applied for the selective, in vitro sensing of miRNA-21 and for the intracellular imaging of miRNA-21 in different cell lines. The LAC12a system is used to image miRNA-21 in different cell cycle phases of MCF-7 cells. Moreover, the LAC12a machinery integrated in cells enables the two-photon laser confocal microscopy-assisted, light-stimulated spatiotemporal, selective activation of the CRISPR-Cas12a miRNA-21 imaging machinery at the single-cell level and the evaluation of relative expression levels of miRNA-21 at distinct cell cycle phases. The method is implemented to map the distribution of cell cycle phases in an array of single cells.

The iPSC secretome is beneficial for in vitro propagation of primary osteoarthritic chondrocytes cell lines

BackgroundOne of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells. MethodsWe added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles. ResultsiPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time. ConclusionOur preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

The CCTδ subunit of the molecular chaperone CCT is required for correct localisation of p150Glued to spindle poles during mitosis

Chaperonin Containing Tailless complex polypeptide 1 (CCT) is a molecular chaperone composed of eight distinct subunits that can exist as individual monomers or as components of a double oligomeric ring, which is essential for the folding of actin and tubulin and other substrates. Here we assess the role of CCT subunits in the context of cell cycle progression by individual subunit depletions upon siRNA treatment in mammalian cells. The depletion of individual CCT subunits leads to variation in the distribution of cell cycle phases and changes in mitotic index. Mitotic defects, such as unaligned chromosomes occur when CCTδ is depleted, concurrent with a reduction in spindle pole-localised p150Glued, a component of the dynactin complex and a binding partner of monomeric CCTδ. In CCTδ-depleted cells, changes in the elution profile of p150Glued are observed consistent with altered conformations and or assembly states with the dynactin complex. Addition of monomeric CCTδ, in the form of GFP-CCTδ, restores correct p150Glued localisation to the spindle poles and rescues the mitotic segregation defects that occur when CCTδ is depleted. This study demonstrates a requirement for CCTδ in its monomeric form for correct chromosome segregation via a mechanism that promotes the correct localisation of p150Glued, thus revealing further complexities to the interplay between CCT, tubulin folding and microtubule dynamics.

UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells

BackgroundThe high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive.MethodExpression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS.ResultWe determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model.ConclusionThese findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.

18‑α‑glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

KRAS is a molecular determinant of platinum responsiveness in glioblastoma

BackgroundKRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis.MethodsLong-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRASG12V plasmids.ResultsHere, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRASG12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype.ConclusionThese findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as ‘actionable target’ of cisplatin-based chemotherapy for glioblastoma.

Microtubule destabilising activity of selected 7-methoxy-2-phenylbenzo[b]furan derivative against primary and metastatic melanoma cells

Herein, we report the results of anticancer screening of two 2-phenylbenzo[b]furan derivatives functionalised at the 3-position with 4-hydroxy-3,5-dimethoxybenzoyl (BF2) or 3,4,5-trimethoxybenzoyl (BF3) against 60 different cancer cell lines. The results confirmed the anticancer potential of the tested compounds against different cancer cell types, especially colon cancer, brain cancer and melanoma. BF3 was defined as the most potent (also as a tubulin polymerisation inhibitor). Its anticancer activity against melanoma cell lines that originated from different stages, i.e., primary skin-derived A375 and metastatic WM9/MDA-MB-435S, was evaluated (as the clinical success of melanoma therapy strictly depends on the disease stage). Moreover, to determine the BF3 mode of action and its effect on cell proliferation, intracellular microtubule networks, cell cycle phase distribution and apoptosis were evaluated. Our study revealed that BF3 inhibited cell proliferation in a dose-dependent manner, with IC50 yielding 0.09 ± 0.01 μM, 0.11 ± 0.01 μM and 0.18 ± 0.05 μM for A375, MDA-MB435S and WM9, respectively. The strong antiproliferative activity of compound BF3 correlated well with its inhibitory effect on tubulin polymerisation. Molecular docking proved that BF3 belongs to the colchicine binding site inhibitors (CBSIs), and experimental studies revealed that it disturbs cell cycle progression leading to G2/M arrest and apoptosis.

Enhancing the therapeutic efficacy of Krestin–chitosan nanocomplex for cancer medication via activation of the mitochondrial intrinsic pathway

Abstract Surgery, chemotherapy, and radiation therapy are all forms of cancer treatment, as well as more recent methods including interventional radiology and immunotherapy. In this study, we synthesize a novel chitosan (CH) nanocomplex (NC)-based polysaccharide Krestin (PSK) for drug delivery. This technique was used to develop PSK@CH@NC. According to the study, PSK@CH@NC had a particle size of around 500 nm, slight polydispersity as observed under a scanning electron microscope, and a strong positive surface charge of 18 mV. Investigation into the in vitro growth inhibition of the MCF-7 cell line after treatment with CH, PSK, and PSK@CH@NC was followed by morphological changes. Compared to other treatment groups, PSK@CH@NC therapy dramatically reduced the fraction of apoptotic cells, cancer cell survival, and proliferation. Fluorescence analysis was used to examine how PSK@CH@NC affected the distribution of cell cycle phases. This study also shows that a promising foundation for creating cancer nanomedicine can be established by employing new polysaccharides.

Circadian gene Per3 promotes astroblastoma progression through the P53/BCL2/BAX signalling pathway

The key circadian genes, Period1(Per1), Period2(Per2), and Period3(Per3), constitute the mammalian Period gene family. The abnormal expression of Per1 and Per2 is closely related to tumor development, but there are few reports on Per3 and tumorigenesis. This study was conducted to determine whether the abnormal expression of Per3 could influence the progression of astroblastoma. The results indicated that the expression level of Per3 was increased in astroblastoma cells, and the high expression of Per3 was correlated with the poor overall survival time of glioma patients. The role of Per3 in astroblastoma cells was then investigated using two approaches: interference and overexpression. The interference of Per3 inhibited astroblastoma cell proliferation by inducing the cell cycle at the S phase. The interference of Per3 inhibited the migration and invasion of astroblastoma cells, while promoted the astroblastoma cell apoptosis and the expression of the apoptosis genes Cleaved-CASP3, P53, and BAX. The overexpression of Per3 promoted proliferation by affecting the S phase distribution of the astroblastoma cell cycle. The overexpression of Per3 promoted the migration and invasion of astroblastoma cells, while inhibited the astroblastoma cell apoptosis and the expression of apoptosis genes Cleaved-CASP3, P53, and BAX. RNA-seq analysis showed that the interference of Per3 in astrocytoma cells resulted in significant changes in the expression levels of 764 genes. Among the differentially expressed genes enriched in apoptosis-related pathways, the interference of Per3 resulted in significant upregulation of MARCKSL1 expression, in contrast to significant downregulation of SFRP4, EPB41L3, and GPC5 expression. Taken together, our results suggest that Per3 appears to be a pro-cancer gene by altering the proliferation, migration, invasion, and apoptosis of astroblastoma cells. As a result, the Per3 gene may be a promising therapeutic target in the treatment of astroblastoma.

Effect of Long-Term Low-Dose Arsenic Exposure on DNA Methylation and Gene Expression in Human Liver Cells.

Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5-10 μM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

SAT632 The Functional Role And Regulatory Mechanism Of FTO M6A RNA Demethylase In Human Uterine Leiomyosarcoma

Abstract Disclosure: Q. Yang: None. A. Al-Hendy: None. Introduction: Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. However, the origin and molecular mechanism underlying driving its clinical and biological behavior remain to be determined. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications contributing to the pathogenesis of uLMS. However, the connectivity between reversible RNA m6A methylation and uLMS remains unclear. In this study, we assessed the role and mechanism of the m6A demethylase fat mass- and obesity-associated protein (FTO) in the pathogenesis of uLMS. Materials and Methods: We performed immunohistochemistry (IHC) and immunoblot analysis to measure the m6A erasers FTO and ALKBH5 protein levels in LMS and adjacent myometrium tissues (n=8) and cell lines. We performed LMS cell proliferation and cell cycle phase distribution analysis. RNA-seq was performed to determine the molecular mechanism underlying m6A eraser inhibition in LMS cells (n=4 for each group). The R package limma+voom was used for this analysis. Hallmark pathway analysis was used to identify enriched pathways in response to FTO inhibition. Real-time PCR was performed to validate the differentially expressed genes. Ingenuity pathway analysis (IPA) was performed to determine the upstream regulators targeting cell cycle-related genes. We used a cutoff −1.5 > fold-change > 1.5 and an adjusted p-value of 0.05. In addition, data were analyzed by a two-tailed unpaired Student’s t-test between any two groups. Results: IHC analysis revealed that m6A demethylases FTO and ALKBH5 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium (p<0.01, H-score). Furthermore, inhibition of FTO with its small, potent inhibitor (Dac51) decreased the uLMS proliferation dose-dependently via cell cycle arrest at G1. Notably, RNA-seq analysis revealed that inhibition of FTO with Dac51 exhibited a significant decrease in the expression of multiple CDK members and a significant increase in the expression of CDKN1A. Furthermore, IPA analysis revealed that transcription factors, including SP1 and E2F members are the upstream regulators targeting CDK members in response to Dac51 treatment. Moreover, Dac51 treatment altered the expression of several epigenetic regulators, including Sirt1 and 2, HDAC10 and SUV39H1, and rewiring of several critical pathways, including inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to suppression of uLMS phenotype. Conclusion: Our findings uncover the anti-uLMS function of a potent FTO inhibitor via reprogramming the oncogenic epitranscriptome. Therefore, targeted inhibition of FTO may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Presentation: Saturday, June 17, 2023

RNA polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation.

Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with β-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation.

Effect of trichostatin A on biological characteristics of side population cells of cervical cancer cell line (HeLa)

Purpose: To investigate the effect of trichostatin A (TSA) on biological characteristics of side population (SP) cells of cervical cancer cell line (HeLa)Methods: Side population (SP) and NSP cells were obtained from 6th generation of primary cervical cancer cells and cervical cancer cell line (HeLa). Cell surface markers (ATP-binding membrane transporter superfamily G member 2 (ABCG2), CD133, CD43, p63, Ki67, multidrug resistance (MDR) as well as cell cycle phase distribution and apoptosis, were determined. SP cells in HeLa were divided into 3 groups that received TSA at doses of 0.01, 0.05, and 0.2 μM. Untreated cells served as control. Cell growth inhibition rates of different dose groups were compared. SP cells in HeLa were divided into simple irradiation group and combined irradiation group TSA (10 % inhibition concentration (IC10) + irradiation, and values of SP cell survival fraction (SF) of the two groups under different irradiation conditions were compared.Results: The content of SP in HeLa cell line was significantly higher than in cervical cancer tissue (p > 0.05). There were no significant differences in expression levels of ABCG2, CD133, CD43, p63, Ki67, and MDR, as well as G0/G1, S, and G2/M phase distributions and apoptosis rate between HeLa cell line and cervical cancer tissue (p > 0.05). On the 3rd, 5th, and 7th day, SF value of SP cells was significantly higher in NSP cells (p > 0.05). With increasing concentration of TSA, SF value of NSP cells gradually decreased, while that of SP cells did not change significantly.Conclusion: Cervical cancer cell line (HeLa) contains SP cells which have biological characteristics of tumor stem cells. Moreover, TSA exerts good cytotoxicity and radio-sensitization effect on SP cells.

PNU-74654 Induces Cell Cycle Arrest and Inhibits EMT Progression in Pancreatic Cancer.

Background and Objectives: PNU-74654, a Wnt/β-catenin pathway inhibitor, has an antiproliferative effect on many cancer types; however, its therapeutic role in pancreatic cancer (PC) has not yet been demonstrated. Here, the effects of PNU-74654 on proliferation and cell cycle phase distribution were studied in PC cell lines. Materials and Methods: The cancer-related molecular pathways regulated by PNU-74654 were determined by a proteome profiling oncology array and confirmed by western blotting. Results: The cell viability and proliferative ability of PC cells were decreased by PNU-74654 treatment. G1 arrest was observed, as indicated by the downregulation of cyclin E and cyclin-dependent kinase 2 (CDK2) and the upregulation of p27. PNU-74654 inhibited the epithelial-mesenchymal transition (EMT), as determined by an increase in E-cadherin and decreases in N-cadherin, ZEB1, and hypoxia-inducible factor-1 alpha (HIF-1α). PNU-74654 also suppressed cytoplasmic and nuclear β-catenin and impaired the NF-κB pathway. Conclusions: These results demonstrate that PNU-74654 modulates G1/S regulatory proteins and inhibits the EMT, thereby suppressing PC cell proliferation, migration, and invasion. The synergistic effect of PNU-74654 and chemotherapy or the exclusive use of PNU-74654 may be therapeutic options for PC and require further investigation.

Modifications of The Human Liver Cancer Cells through microRNA-145-Mediated Targeting of CDCA3.

Owing to the lethality of liver cancer, it is considered as one of the devastating types of cancers across the globe. Consistently, the study was designed to elucidate the role and to explore the therapeutic implications of miR-145 in human liver cancer. In the current experimental study, gene expression was determined by RT-PCR analysis. Transfection of cancer cells was carried out using Lipofectamine 2000. The cell proliferation of liver cancer cells was estimated by MTT assay. Clonogenic assay was performed for analysis of colony forming potential of cancer cells. Flow cytometry was done to analyze the cell cycle phase distribution of cancer cells. Transwell chamber assay was performed to assess the motility of cancer cells. Western blotting was done to estimate the expression levels of proteins. Dual luciferase assay was performed for interaction analysis of miR-145 with CDCA3. The miR-145 expression was found to be downregulated in liver cancer cells. The transfection mediated overexpression of miR-145 inhibited the cancer cell proliferation and when miR-145 inhibitor was transfected, cancer cells showed higher proliferation rates. Enrichment of miR-145 levels led to cell cycle arrest at G2/M phase by inhibiting cyclin B1. miR-145 also restricted the migration and invasion of cancer cells. CDCA3 was shown to be the intracellular target of miR-145 and it was found that the inhibitory effects of miR-145 were modulated through CDCA3, intracellularly. The current study clearly revealed that there is a need to investigate the regulatory role of different molecular entities like microRNAs in cancer development to better understand mechanics behind this pathogenesis and design more effective combating strategies against cancer.

Effects on cell cycle progression and cytoskeleton organization of five Bothrops spp. venoms in cell culture-based assays

Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.