G1 Cell Research Articles

Resistance gene-guided genome mining reveals the roseopurpurins as inhibitors of cyclin-dependent kinases.

With the significant increase in the availability of microbial genome sequences in recent years, resistance gene-guided genome mining has emerged as a powerful approach for identifying natural products with specific bioactivities. Here, we present the use of this approach to reveal the roseopurpurins as potent inhibitors of cyclin-dependent kinases (CDKs), a class of cell cycle regulators implicated in multiple cancers. We identified a biosynthetic gene cluster (BGC) with a putative resistance gene with homology to human CDK2. Using targeted gene disruption and transcription factor overexpression in Aspergillus uvarum, and heterologous expression of the BGC in Aspergillus nidulans, we demonstrated that roseopurpurin C (1) is produced by this cluster and characterized its biosynthesis. We determined the potency, specificity, and mechanism of action of 1 as well as multiple intermediates and shunt products produced from the BGC. We show that 1 inhibits human CDK2 with a Kiapp of 44 nM, demonstrates selectivity for clinically relevant members of the CDK family, and induces G1 cell cycle arrest in HCT116 cells. Structural analysis of 1 complexed with CDK2 revealed the molecular basis of ATP-competitive inhibition.

TMIC-26. TAMS AND HYPOXIA ENHANCE GLIOBLASTOMA STEM-LIKE CELL ENRICHMENT AND MAY IMPACT RADIORESISTANCE

Abstract The dismal survival rates of glioblastoma (GBM) patients are due, at least in part, to therapeutic resistance of GBM stem-like cells (GSCs) and their ability to repopulate recurrent tumors. Our goal was to determine if tumor-associated macrophages (TAMs) or hypoxia enhance the enrichment of GSCs in a manner that could impact radioresistance. First, we established GBM-brain cortical organoids (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurospheres with BCOs for 14 days and observed a tumor take rate of 100%. Next, we evaluated whether hypoxia enhanced TAM migration into GBM-BCOs by incorporating THP-1 cells into GFP-GBM-BCOs and incubating them for 24 h under normoxic or hypoxic conditions. We found that hypoxia significantly increased THP-1 cell influx into GBM-BCOs. Further, we investigated whether TAMs influenced the enrichment of GSCs in GBM neurospheres and in GBM-BCOs. We found that co-culture of THP-1 cells with either GBM neurospheres or with GBM-BCOs enhanced the CD133+ population after 72 h hypoxia, correlating with our IF studies of human GBM samples that show increased CD133+ and IBA1+ cells in hypoxic peri-necrotic zones compared to non-necrotic regions. Next, we established Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), an advanced methodology for monitoring cell cycle phases for investigating the role of hypoxia and radiation on GBM. Flow cytometry analysis of patient-derived GBM cells revealed that the fraction of G1 cells decreased and the fraction of G2/M cells increased 24 h after 3 Gy radiation compared to non-irradiated cells in normoxia. In contrast, GBM cells exposed to hypoxia do not show such cell cycle shifts after radiation and have an enriched population of cells in G0 in both radiated and non-irradiated cells. Our results indicate that hypoxia and TAMs play critical roles in GSC enrichment and may impact radioresistance.

CCRG-03. KAT5 REGULATES DEVELOPMENTAL TRANSITIONS AND CELLULAR HETEROGENEITY IN AN IN VIVO MODEL OF GLIOBLASTOMA

Abstract In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we previously performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of GBM G0 ingress/egress. Here, we show in an in vivo GSC-derived orthotopic xenograft model that KAT5 regulates transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. We show that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics. With regard to chromatin regulation, we show that loss of KAT5 activity alters sites of mixed epigenetic valency (which have both activating and repressive marks in bulk tumor populations) to promote emergence of OPC/RG G0-like populations in tumors. Our data reveal KAT5-associated transcriptional and epigenetic changes in tumors, which indicate that KAT5 activity biases cell state distribution away from OPC and RG-like subpopulations. In addition, loss of KAT5 resulted in a less invasive phenotype and prolonged survival of mice harboring orthotopic xenografts.

DNAR-13. TARGETING THE DNA DAMAGE RESPONSE IN GLIOBLASTOMA PROPAGATING CELLS: FROM MOLECULAR MECHANISMS TO PRECISION MEDICINE

Abstract Glioblastoma (GBM) is the most aggressive and lethal primary tumor of the central nervous system (CNS). Despite surgical resection and a combination of radiotherapy and alkylation chemotherapy, the median survival for patients with newly diagnosed GBM is only ~ 14 months. Among the many challenges confronting clinicians are subpopulations of tumor-propagating cells named GBM Stem-like Cells (GSCs), which have been proposed to drive treatment resistance and foster tumor relapse. At the mechanistic level, GSCs have been shown to display constitutive activation of the DNA damage response (DDR) due to chronic replication stress (RS), which enhances their ability to repair the DNA lesions induced by chemoradiation. Thus, developing new therapeutic strategies against glioblastoma is of paramount importance. Cell cycle regulation and DNA damage response are two crucial molecular networks altered in most cancer types. In this context, we have previously identified a DNA repair and cell cycle gene expression signature that results in the classification of GBM tumors in two major groups, G1 and G3, which differ in the expression of survival and proliferation pathways. Using bioinformatics analysis of the GLASS consortium database, we have created an exhaustive oncopanel of the molecular alterations defining the G1 and G3 groups. Furthermore, using G1- and G3-patient-derived GSC cell lines as experimental models, we have identified crucial differences in the mechanisms orchestrating the cell cycle, DDR, and treatment resistance between G1 and G3 cells. Finally, we have also identified group-specific therapeutic strategies combining repositioned FDA-approved drugs with the inhibition of crucial DDR factors. Our long-term goals are i) to understand the molecular basis behind the differential response to DNA damage and replication stress developed by G1 and G3 cells and ii) to propose group-specific, DDR-based combination therapies that prevent treatment resistance and promote long-lasting treatment.

DEPDC1B enhances malignant phenotypes of multiple myeloma through upregulating CCNB1 and inhibiting p53 signaling pathway

The identification and investigation of key molecules involved in the pathogenesis of multiple myeloma (MM) hold paramount clinical significance. This study primarily focuses on elucidating the role of DEPDC1B within the context of MM. Our findings robustly affirm the abundant expression of DEPDC1B in MM tissues and cell lines. Notably, DEPDC1B depletion exerted inhibitory effects on MM cell proliferation and migration while concurrently facilitating apoptosis and G2 cell cycle arrest. These outcomes stand in stark contrast to the consequences of DEPDC1B overexpression. Furthermore, we identified CCNB1 as a putative downstream target, characterized by a co-expression pattern with DEPDC1B, mediating DEPDC1B's regulatory influence on MM. Additionally, our results suggest that DEPDC1B knockdown may activate the p53 pathway, thereby impeding MM progression. To corroborate these in vitro findings, we conducted in vivo experiments that further validate the regulatory role of DEPDC1B in MM and its interaction with CCNB1 and the p53 pathway. Collectively, our research underscores DEPDC1B as a potent promoter in the development of MM, representing a promising therapeutic target for MM treatment. This discovery bears significant implications for future investigations in this field.

CDCA2 Is a Predictive Marker and Mediator of Bortezomib Response in Diffuse Large B-Cell Lymphoma

Objective: In recent years randomized phase III clinical trials have attempted to improve first-line R-CHOP treatment of diffuse large B-cell lymphoma (DLBCL) through addition or substitution of drugs. Bortezomib treatment has shown significant clinical efficacy in other B-cell malignancies, and the REMoDL-B trial testing R-CHOP vs. RB-CHOP in DLBCL patients stratified by cell of origin revealed at 60 months follow-up that ABC and molecular high-grade patients significantly benefitted in clinical outcome. This study aims at identifying predictive markers of bortezomib response combined with functional investigation of their biological impact in DLBCL irrespective of molecular subtypes. Methods: Data from the REMoDL-B trial including 450 patients in each treatment arm was retrospectively analyzed. We fitted a multivariate Cox regression model testing all expressed genes with overall survival as outcome and an interaction term between gene expression and treatment arms (R-CHOP and RB-CHOP) as covariates, including their main effects. For the top candidate, CDCA2, survival was visualized using Kaplan-Meier analysis of patients stratified by median expression. Functional examination of CDCA2 was performed by applying CRISPR/Cas9 genome editing. Knockout of CDCA2 was conducted in two DLBCL cell lines, representing ABC and GCB cell of origin, and effect on proliferation, cell cycle distribution, and drug response was examined in MTS-assays and by flow cytometry. A DLBCL xenograft mouse model was established with CDCA2 knockout and control DLBCL cells, respectively, and time to tumor formation and death was monitored and tumors molecularly characterized. Results: DLBCL patients with high expression of CDCA2 have superior outcome when treated with RB-CHOP in comparison to R-CHOP (p=0.0097), whereas no difference in outcome was observed for patients with low CDCA2 expression (p=0.086) (Figure 1), supporting high expression of CDCA2 as a predictive marker of bortezomib response irrespective of cell of origin subclassification. Knockout of CDCA2 decreased DLBCL cell proliferation rate and affected the cell cycle transition between the G1 and S-phase, resulting in an increased percentage of cells in G1 compared to the scramble control. Bortezomib dose-response analysis revealed reduced sensitivity in CDCA2 knockout cells compared to the control (Figure 2A). In addition, CDCA2 knockout cells were also less sensitive to carfilzomib, a 2 nd generation proteasome inhibitor. In combinatory drug experiments comparing CHOP and B-CHOP, CDCA2 knockout cells demonstrated higher relative resistance compared to controls cells when exposed to B-CHOP than CHOP (Figure 2B+C), illustrating CDCA2 to be cardinal and specifically mediating the bortezomib response. In DLBCL xenograft models, CDCA2 knockout tumors displayed reduced growth rate in comparison to control tumors. Due to the substantial tumor burden, control DLBCL xenografted mice had shorter survival time than CDCA2 knockout DLBCL mice (Figure 2D). Conclusion: DLBCL patients with high expression levels of CDCA2 benefitted the most from addition of bortezomib to R-CHOP. In functional studies, the loss of CDCA2 increased cellular resistance to bortezomib in both single- and combinational drug studies.

Narazaciclib, a Differentiated CDK4/6 Antagonist, Prolongs Cell Cycle Arrest and Metabolomic Reprogramming, Enabling Restoration of Ibrutinib Sensitivity in Btki-Resistant Mantle Cell Lymphoma

Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL. We compared the efficacy and safety profiles of narazaciclib with three health authority-approved CDKi (palbociclib, abemaciclib or ribociclib) in association with covalent (ibrutinib, acalabrutinib) and non-covalent (pirtobrutinib, ARQ-531) BTKi, across a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We integrated RNA sequencing and gene set enrichment analysis (GSEA) coupled to phospho-proteomics and kinase-substrate enrichment analysis (KSEA) to unravel the molecular bases of BTKi-CDKi drug interaction in MCL. CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, RT-PCR and western blot approaches followed by Seahorse-mediated metabolic assays were carried out for validation purposes. Finally, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in three immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft models. We found that narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC 50: 3.61 ± 2.1 µM), regardless of the sensitivity of these to ibrutinib. Although there was no correlation between CDKi sensitivity and direct repression of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response upon narazaciclib treatment. This feature was linked to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second-generation acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTKi-sensitive and BTKi-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome, although similar features were found when substituting narazaciclib with abemaciclib. The integration of transcriptomic and phospho-proteomic synergistic signatures further revealed that narazaciclib-ibrutinib combo modulated signatures related to DNA repair, P53 signaling, and glycolytic activity. Accordingly, in this model, narazaciclib/ibrutinib-exposed cells underwent a metabolic switch from glycolytic to oxidative phosphorylation (OxPhos) phenotype accompanied by intracellular accumulation of mitochondria and increased mtDNA copy number. This switch appeared to be specific to MCL cells rendered refractory to ibrutinib upon activation of alternative NFkB signaling, as it was not found in MCL-BTKmut cells. In vivo, while narazaciclib single agent achieved a moderate TGI in the CAM model, the narazaciclib/ibrutinib combo reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into host bone marrow, with no detectable toxicity. In conclusion, our findings demonstrate that narazaciclib is safe and effective as a single agent in preclinical models of MCL, including BTKi-resistant cases. Its combination with ibrutinib achieved a synergistic tumoricidal effect in vitro and in vivo, accelerating cell cycle blockade and reverting the metabolic reprogramming characterizing MCL refractoriness to BTKi therapy.

Harnessing Btki Therapy By CDK4/6i Control of T Effector Memory Cells and T Cell Surveillance in Mantle Cell Lymphoma

Drug resistance remains a formidable challenge in mantle cell lymphoma (MCL). Cell cycle dysregulation driven by aberrant Cyclin D1 and CDK4 expression is a hallmark for MCL, providing a rationale for targeting the cell cycle in MCL therapy. We have demonstrated in preclinical studies that inhibition of CDK4/6 not only induces early G1 cell cycle arrest, but also reprograms MCL cells for cytotoxic killing. On this basis, we investigated if inhibition of cyclin D1/CDK4 with palbociclib (CDK4/6 inhibitor) could reprogram recurrent MCL to deepen and prolong the ibrutinib (BTK inhibitor) response in a phase I clinical trial, and the resistance mechanism by longitudinal genomic analysis of sequential samples from individual patients in the context of the clinical response. Palbociclib was administered to MCL patients on days 1-21 of a 28-day cycle; ibrutinib was given continuously. For longitudinal genomic analysis, sequential tissue and blood specimens from 27 evaluable MCL patients before and during therapy, and on progression were collected. Single cell RNA-seq (scRNA-seq) of PBMC or the monocytic fractions from bone marrow and lymph node (53 samples) was performed using a unique in house MCL cell-specific library. PBMC from normal donors (n=4) and untreated MCL patients (n=4) served as controls. The data were then subject to multiplex analysis with whole transcriptome sequencing and whole exome sequencing of MCL cells isolated from the same specimens, as well as flowcytometry in conjunction with IHC. Total numbers of MCL and immune cells in each specimen were deduced by integrating complete blood counts (CBC) with differential with scRNA-seq or flowcytometry. CDK4/6 Inhibition appears to deepen and prolong the BTKi response, with a CR rate of 42% and 5 patients (2 CR and 3 PR) remained on therapy for ~ 9 years. Longitudinal scRNA-seq (210,000 cells) revealed that MCL cells comprise 4 major transcriptomically distinct clusters. Cluster 1 (C1) resembles quiescent normal B cells; C2 mimics hyper-activated B cells that are enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative. RNA velocity analysis further indicates that having progressed to late G1 after evading CDK4/6 inhibition, MCL cells in C2 are in transit to C4. By contrast, C3 is a terminal node of MCL cells arrested in late G1 with elevated BCL2 expression Primary resistance and progression on therapy were associated with a marked expansion of either long-live non-proliferating C3 MCL cells, or C2 MCL cells that fuel the proliferating C4 MCL cells. Resistance was also associated with a profound reduction in MHC I and MHC II expression on MCL cells in some patients. Moreover, concurrent with disease progression, CD8+ T and CD+T cells were rapidly depleted by different mechanisms ( Figure 1). CD8+ T cells were proliferating and eliminated by exhaustion, as evidenced by the marked expression of LAG3 TIGIT and TIM3, and a failure to maintain CD8+ T effector memory cells. By contrast, CD4+ T cells were not proliferating and appeared die from glucose starvation. Collectively, these data suggest that T cell maintenance and surveillance is pivotal in sustaining a durable clinical response to combined inhibition of CDK4/6 and BTK. Guided by these discoveries, we have now restored BTKi sensitivity ex vivo in MCL cells from resistant patientsby targeting patient-specific expansion of C2 or C3 in vivo. In one resistant patient, this led to the maintenance of CD4+ and CD8+ T effector memory cells and a CR in response to venetoclax plus BTKi for nearly 3 years and continuing. In summary, by integrated longitudinal scRNA-seq analysis of a hypothesis-driven therapy, we have provided the first evidence that 1) MCL cells comprise 4 major transcriptomically distinct clusters; 2) resistance to CDK4/6i is associated with expansion of C2 that fuels the proliferating C4 MCL cells, or the long-lived C3 MCL cells with elevated BCL2 expression; 3) CDK4/6i harnesses BTKi response by promoting the maintenance of effector memory T cells; 4) combined inhibition of BCL2 and BTK overrides resistance to CDK4/6i and BTKi, leading to a durable clinical response; and 5) T cell surveillance is pivotal for a durable response to CDK4/6, BTK or BCL2 inhibition; implicating genome-guided combination therapy to overcome therapy resistance in MCL.

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest

CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.

Harnessing Nature's Gifts: Salix nigra and Its Potential for Combating Hepatitis C Virus (HCV).

Hepatitis C virus (HCV) causes various liver complications, including fibrosis, cirrhosis, and steatosis, and finally progresses toward hepatocellular carcinoma (HCC). The current study aimed to explore the antiviral activity of the traditional Pakistani medicinal plant Salix nigra (S. nigra) known as black willow against the hepatitis C virus (HCV). The anti-HCV activity of S. nigra was established against stable Hep G2 cell lines expressing the HCV NS3 gene. Various plant-derived compounds with anti-HCV activity were identified, making phytotherapy a promising alternative to conventional treatments due to their cost-effectiveness and milder side effects. The two extraction methods (Maceration and Soxhlet) and four solvents (n-hexane, methanol, ethyl acetate, and water) were used to obtain crude extracts from S. nigra. Cytotoxicity testing showed that methanol (CC50 25 μg/mL) and water (CC50 30 μg/mL) extracts were highly toxic, while ethyl acetate and n-hexane (CC50 > 200 μg/mL) extracts were nontoxic at low concentrations (10-50 μg/mL), making them suitable for further anti-HCV investigations. Stable transfection of the NS3 gene was successfully performed in Hep G2 cells, creating a cellular expression system for studying virus-host interaction. The ethyl acetate extract of S. nigra exhibited significant inhibition of NS3 gene expression (mRNA and protein levels). The phytochemical analysis of S. nigra was also performed using the high-performance liquid chromatography (HPLC) technique. The phytochemical analysis identified several polyphenolic substances in the extracts of S. nigra. Our results concluded that the extracts of S. nigra have significantly reduced the expression of the NS3 gene at mRNA and protein levels. These findings contribute to the global efforts to combat hepatitis C by offering plant-based treatment options for HCV management.

A Combination of CPI-0610 and SAHA Induces Apoptosis through STAT3 and p38 Signalling Pathways in Diffuse Large B-cell Lymphoma Cells.

Although immunotherapies have greatly improved diffuse large B-cell lymphoma (DLBCL) prognosis, a proportion of patients remain to be relapsed or refractory. Therefore, the identification of novel therapeutic targets and drugs is urgently required. Inhibition of the bromodomain and extra-terminal (BET) proteins has been a promising therapeutic strategy for various haematologic cancers. CPI-0610 is a potent and selective BET inhibitor. The effects of CPI-0610 in DLBCL cells have not been reported yet. The aim of this study was to assess the effects of CPI-0610 in DLBCL and its underlying mechanisms. DLBCL cells were treated with CPI-0610, followed by measuring cell viability, cell cycle, apoptosis, autophagy, and specific cell signaling pathways. Moreover, immunodeficient mice were engrafted with SUDHL2 cells and then treated with CPI-0610 for analysis of tumor burden. We also analyzed the synergistic effect of CPI-0610 with histone deacetylase inhibitor suberoylanilide hydroxamic acid. The present study demonstrated that CPI-0610 displayed cell cytotoxicity by arresting the G1 cell cycle and inducing endogenous and exogenous apoptotic pathways. Additionally, CPI-0610 decreased BRD4 and c-Myc expressions and affected MAPK, JAK/STAT, and AKT signalling pathways in human DLBCL cells. An in vivo experiment exhibited that CPI-0610 decreased the primary tumour growth of the DLBCL xenograft model. Furthermore, the use of CPI-0610 in combination with suberoylanilide hydroxamic acid exhibited a specific synergistic effect in inducing apoptosis through the regulation of STAT3 and p38. Targeting BET may be an effective therapeutic strategy and potentiated by a combination with histone deacetylase inhibition in DLBCL.

Epithelial-mesenchymal transition: an organizing principle of mammalian regeneration.

Introduction: The MRL mouse strain is one of the few examples of a mammal capable of healing appendage wounds by regeneration, a process that begins with the formation of a blastema, a structure containing de-differentiating mesenchymal cells. HIF-1α expression in the nascent MRL wound site blastema is one of the earliest identified events and is sufficient to initiate the complete regenerative program. However, HIF-1α regulates many cellular processes modulating the expression of hundreds of genes. A later signal event is the absence of a functional G1 checkpoint, leading to G2 cell cycle arrest with increased cellular DNA but little cell division observed in the blastema. This lack of mitosis in MRL blastema cells is also a hallmark of regeneration in classical invertebrate and vertebrate regenerators such as planaria, hydra, and newt. Results and discussion: Here, we explore the cellular events occurring between HIF-1α upregulation and its regulation of the genes involved in G2 arrest (EVI-5, γH3, Wnt5a, and ROR2), and identify epithelial-mesenchymal transition (EMT) (Twist and Slug) and chromatin remodeling (EZH-2 and H3K27me3) as key intermediary processes. The locus of these cellular events is highly regionalized within the blastema, occurring in the same cells as determined by double staining by immunohistochemistry and FACS analysis, and appears as EMT and chromatin remodeling, followed by G2 arrest determined by kinetic expression studies.

Near‐Infrared Emission‐Cyanine 5.5 and Doxorubicin Loaded Magnetite Nanoparticles for Dual In Vivo Magnetic Resonance and Biodistribution Imaging

AbstractThe in vivo imaging of the drug distribution during cancer treatment plays an important role in helping physicians and pharmacists to monitor the progress of treatment and improve the effectiveness of therapy. Our goal in this work was to prepare a multifunctional nano drug delivery system that not only co‐loaded iron oxide nanoparticles (Fe3O4 NPs) and Doxorubicin (Dox) but also attached the near‐infrared fluorescent agent Cyanine 5.5 based on poly(lactide)‐tocopheryl polyethylene glycol 1000 succinate (PLA‐TPGS) copolymer. Cyanine 5.5 was attached to the surface Fe3O4 NPs via the NH−CO chemical bond, followed by co‐loading of Dox onto the PLA‐TPGS copolymer by the emulsion solvent evaporation method. The structural, morphological, dimensional, stability, and optical‐magnetic properties of the as‐synthesized nanosystem were fully characterized. From the drug‐released study, the prolonged release of Dox accelerated with increasing acidic conditions (at pH 5.0 and 6.5), while a slow release was observed at pH 7.4. Through the MTT assay, the nanosystem had cellular toxicity against Hep−G2 and HeLa cell lines with low IC50 values of 0.42 and 0.78 μg.mL−1 in terms of Dox concentration, respectively. At a magnetic field of 7 T, this nanosystem also exhibited a high r2/r1 ratio of 164.5 and a strong contrast magnetic resonance (MR) image in the liver. In vivo biodistribution studies revealed that the highest fluorescence intensity achived in the liver after 6 h, and the nanosystem was eliminated from the body after at least 24 h. Collectively, these results demonstrated that the PLA‐TPGS‐Fe3O4‐Cyanine 5.5‐Dox nanosystem is a good candidate for simultaneously increasing contrast for MR and near‐infrared fluorescence imaging.

Cytotoxic Indole Diterpenoids from a Sphagneticola trilobata-Derived Fungus Aspergillus sp. PQJ-1.

Two new indole diterpene derivatives, 5S-hydroxy-β-aflatrem (1) and 14R-hydroxy-β-aflatrem (2), along with one known analogue, 14-(N,N-dimethl-L-valyloxy)paspalinine (3), were isolated from the fermentation broth of the fungus Aspergillus sp. PQJ-1 derived from Sphagneticola trilobata. The structures of the new compounds were elucidated from spectroscopic data and ECD spectroscopic analyses. All the compounds (1-3) were evaluated for their cytotoxicity against A549, Hela, Hep G2, and MCF-7 cell lines. Compounds 1 and 2 exhibited selective inhibition against Hela cells. Further studies showed that 1 significantly induced apoptosis and suppressed migration and invasion in Hela cells. Moreover, 1 could up-regulate pro-apoptotic genes BAX and Caspase-3 and down-regulate anti-apoptotic genes Bcl-xL and XIXP.

Impact of CRISPR/Cas9-Mediated CD73 Knockout in Pancreatic Cancer.

Pancreatic cancer is among the cancers with the highest mortality rates. Most of the patients are found to have advanced cancer, losing the chance of surgical treatment, and there is an urgent need to find new treatment methods. Targeted therapy for specific genes that play a key role in cancer is now an important means to improve the survival rate of patients. We determined that CD73 is highly expressed in pancreatic cancer by flow cytometry and qRT-PCR assays combined with bioinformatics techniques. Application of CRISPR/Cas9 technology to knockout CD73 in human and murine cell lines, respectively, revealed that CD73 inactivation inhibited cell growth and migration and induced G1 cell cycle arrest. We also found that CD73 deletion inhibited the ERK/STAT3 pathway and activated the E-cadherin pathway. In addition, a CRISPR/Cas9 protein kinase library screen was performed and identified Pbk, Fastk, Cdk19, Adck5, Trim28, and Pfkp as possible genes regulating CD73.

Small molecule targeting of the p38/Mk2 stress signaling pathways to improve cancer treatment

PurposeAlthough a long-term goal of cancer therapy always has been the development of agents that selectively destroy cancer cells, more recent trends have been to seek secondary agents that sensitize cancer cells to existing treatment regimens. In this regard, the present study explored the possibility of using small molecule inhibitors of p38MAPK/MK2 stress signaling pathways as potential agents to enhance the sensitivity of cancer cells with abrogated G1 checkpoint to the DNA damaging agent etoposide by specifically targeting the DNA damage-induced G2 cell cycle checkpoint.MethodsWe have applied CCK8 and FACS-based viability assays and cell cycle analysis to investigate the effect of small molecules SB203580 and MK2.III on the sensitivity of small cell lung cancer cells (SCLC) that lack the G1 checkpoint to the DNA damaging agent Etoposide when used in combination. We have also assessed the effectiveness of combination chemotherapy on tumor xenograft suppression with etoposide and MK2.III in immunosuppressed mice. In addition, additional CCK8 cell viability analysis of the MDA-MB-231 breast cancer cell line, and SW620, and SW480 colorectal cancer cell lines was performed.ResultsResults suggest that etoposide produces a profound effect on the cell cycle profile of cells in a manner that is consistent with the degree of cell viability that is seen using the viable cell assay. Results of the co-treatment experiments revealed that the p38/MK2 kinase inhibitors SB203580 and MK2.III both enhanced the DNA-damaging effects of etoposide on NCI-H69 cell viability in vitro. Results revealed that in vivo MK2.III was able to act as a chemosensitizer when used in combination with etoposide making NCI-H69 lung cancer cells sensitive to chemotherapeutic drug by 45% compared to single usage of the drug. We also report that MK2.III sensitizes metastatic cell lines SW-620 and MDA-MB-231 to etoposide but does not increase the sensitivity of non-metastasizing SW-480 colorectal cells to DNA damaging agent in vitro.ConclusionFindings reported in this study provide evidence that specific inhibitors of MK2 may indeed improve overall cancer therapy; however, their effectiveness depends on cell types.

Formulation, characterization and evaluation of gelatin-syringic acid/zinc oxide nanocomposite for its effective anticancer, antioxidant and anti-inflammatory activities

BackgroundHepatocellular carcinoma (HCC) is the most typical form of liver cancer. Apart from modern therapies, various natural chemical constituents have been tested for the treatment of HCC. However, the usage of the latter has declined due to their low bioavailability and stability. ObjectivesConsidering the above drawback’s, in this study, we describe a gelatin-syringic acid/ zinc oxide (ZnO) nanocomposite for liver cancer treatment, which showed antioxidant, anti-inflammatory and anticancer activities. MethodsThe hydrothermal method was used to synthesize ZnO nanoparticle and it was encapsulated with gelatin-syringic acid by coacervation technique. The nanocomposites were characterized by UV spec, FTIR, SEM, XRD, EDX, and DLS. The drug release profile of nanocomposite was studied by dialysis bag method, which exhibits a sustained drug release. ResultsThe antioxidant ability of nano-composite was studied by performing an ABTS and DPPH assay and the IC50 was recorded as 76.5 and 47.63 µg/mL, respectively. The anti-inflammatory potential was studied by assessing the ability of nanocomposite on denaturation of protein and the results exhibited a dose-dependent inhibition. The acute toxicity of nanocomposite tested on zebrafish liver and heart showed that it is non-toxic. Moreover, the nanocomposite inhibits the Hep G2 cell viability with an increasing concentration and it increases oxidative stress caused by the mitochondrial damage, which leads to cell death. ConclusionBased on the findings of the present study, gelatin-syringic acid/ZnO nanocomposite has been shown to possess antioxidant, and anti-inflammatory properties, and thus can be used against HCC.

ATR activation by Cr-DNA damage is a major survival response establishing late S and G2 checkpoints after Cr(VI) exposure

Inhalation exposure to hexavalent chromium is known to cause lung cancer and other pulmonary toxicity. Cellular metabolism of chromium(VI) entering cells as chromate anion produces different amounts of reactive Cr(V) intermediates and finally yields Cr(III). Direct reduction of Cr(VI) by ascorbate (Asc), the dominant metabolic reaction in vivo but not in standard cell cultures, skips production of Cr(V) but still permits extensive formation of Cr-DNA damage. To understand the importance of different forms of biological injury in Cr(VI) toxicity, we examined activation of several protein- and DNA damage-sensitive stress responses in human lung cells under Asc-restored conditions. We found that Asc-restored cells suppressed upregulation of oxidant-sensitive stress systems by Cr(VI) but showed a strong activation of the apical DNA damage-responsive kinase ATR. ATR signaling was triggered in late S phase and persisted upon entry of cells into G2 phase. Inhibition of ATR prevented the establishment of late-S and G2 cell cycle checkpoints and did not lead to a compensatory activation of a related kinase ATM. Inactivation of ATR also strongly impaired viability of Cr(VI)-treated lung cells including stem-like cells and revealed a significant formation of toxic Cr-DNA damage at low Cr(VI) doses. Our findings identified a major Cr(VI) resistance mechanism involving sensing of Cr-DNA damage by ATR in late S phase and a subsequent establishment of protective cell cycle checkpoints.

G1 Dynamics at the Crossroads of Pluripotency and Cancer.

G1 cell cycle phase dynamics are regulated by intricate networks involving cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors, which control G1 progression and ensure proper cell cycle transitions. Moreover, adequate origin licensing in G1 phase, the first committed step of DNA replication in the subsequent S phase, is essential to maintain genome integrity. In this review, we highlight the intriguing parallels and disparities in G1 dynamics between stem cells and cancer cells, focusing on their regulatory mechanisms and functional outcomes. Notably, SOX2, OCT4, KLF4, and the pluripotency reprogramming facilitator c-MYC, known for their role in establishing and maintaining stem cell pluripotency, are also aberrantly expressed in certain cancer cells. In this review, we discuss recent advances in understanding the regulatory role of these pluripotency factors in G1 dynamics in the context of stem cells and cancer cells, which may offer new insights into the interconnections between pluripotency and tumorigenesis.

Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs.

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.