G1 Cell Research Articles

Deficiency in homologous recombination is associated with changes in cell cycling and morphology in Saccharomyces cerevisiae

Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated internally by chemicals and enzymes, in the absence of exogenous agents, though the sources and consequences of such endogenously generated DSBs remain poorly understood. In the current study, we have investigated the impact of reduced recombinational repair of endogenous DSBs on stress responses, cell morphology and other physical properties of S. cerevisiae (budding yeast) cells. Use of phase contrast and DAPI-based fluorescence microscopy combined with FACS analysis confirmed that recombination-deficient rad52 cell cultures exhibit chronically high levels of G2 phase cells. Cell cycle phase transit times during G1, S and M were similar in WT and rad52 cells, but the length of G2 phase was increased by three-fold in the mutants. rad52 cells were larger than WT in all phases of the cycle and displayed other quantifiable changes in physical characteristics. The high G2 cell phenotype was abolished when DNA damage checkpoint genes, but not spindle assembly checkpoint genes, were co-inactivated with RAD52. Several other RAD52 group mutants (rad51, rad54, rad55, rad57 and rad59) also exhibited the high G2 cell phenotype. The results indicate that recombination deficiency leads to accumulation of unrepaired DSBs during normal mitotic growth that activate a major stress response and produce distinct changes in cellular physiology and morphology.

Spectroscopic, thermal, and anticancer investigations of new cobalt(II) and nickel(II) triazine complexes

ABSTRACT. The condensation of thiosemicarbazide with oxazolidinone produced 5-benzylidene-3-(4-chloro-phenyl)-6-oxo-5,6-dihydro-1H-[1,2,4]triazine-2-carbothioic acid amide (HL1). Co(II) and Ni(II) ions were reacted with the HL1 ligand. Elemental analysis and molar conductivity, along with mass, 1H-NMR, IR, UV-Vis, and X-ray powder diffraction spectral examinations, were used to reveal the chemical structure of the synthesized complexes. The stoichiometry of the complexes was determined to be 1:2 (metallic: moiety) using analytical, spectroscopic, and thermal data. The thermal properties of the complexes were explored using thermogravimetric (TG-DTG/DTA) methods, as well as the decomposition stages. Several kinetic thermodynamic parameters such as free activation energy (ΔG*), activation enthalpy (ΔH*), activation entropy (ΔS*), pre-exponential factor (A), and the activation energy (E*) were estimated. Inhibitory effects towards colon carcinoma cells (HCT cell line), hepatocellular carcinoma (Hep G2 cell line), and breast carcinoma (MCF-7 cell line) were verified using various concentrations of the samples. A colorimetric method was used to determine the cell viability percent in comparison to doxorubicin drug as a control.
 
 KEY WORDS: 1,2,4-Triazines, Thiosemicarbazide, Thermogravimetric analysis, Non-electrolytes, Anticancer
 Bull. Chem. Soc. Ethiop. 2023, 37(5), 1151-1162. 
 DOI: https://dx.doi.org/10.4314/bcse.v37i5.8

Extracts from Cnidium monnieri (L.) Cusson Induces Apoptosis and G1 Cell Cycle Arrest through Reducing COX-2 Expression by Regulating the Akt/GSK-3β Signaling Pathways

Extracts from <i>Cnidium monnieri</i> (L.) Cusson Induces Apoptosis and G1 Cell Cycle Arrest through Reducing COX-2 Expression by Regulating the Akt/GSK-3β Signaling Pathways

Mus musculus papillomavirus 1 E8^E2 represses expression of late protein E4 in basal-like keratinocytes via NCoR/SMRT-HDAC3 co-repressor complexes to enable wart formation in vivo.

High-risk human papillomaviruses (PV) account for approximately 600,000 new cancers per year. The early protein E8^E2 is a conserved repressor of PV replication, whereas E4 is a late protein that arrests cells in G2 and collapses keratin filaments to facilitate virion release. While inactivation of the Mus musculus PV1 (MmuPV1) E8 start codon (E8-) increases viral gene expression, surprisingly, it prevents wart formation in FoxN1nu/nu mice. To understand this surprising phenotype, the impact of additional E8^E2 mutations was characterized in tissue culture and mice. MmuPV1 and HPV E8^E2 similarly interact with cellular NCoR/SMRT-HDAC3 co-repressor complexes. Disruption of the splice donor sequence used to generate the E8^E2 transcript or E8^E2 mutants (mt) with impaired binding to NCoR/SMRT-HDAC3 activates MmuPV1 transcription in murine keratinocytes. These MmuPV1 E8^E2 mt genomes also fail to induce warts in mice. The phenotype of E8^E2 mt genomes in undifferentiated cells resembles productive PV replication in differentiated keratinocytes. Consistent with this, E8^E2 mt genomes induced aberrant E4 expression in undifferentiated keratinocytes. In line with observations for HPV, MmuPV1 E4-positive cells displayed a shift to the G2 phase of the cell cycle. In summary, we propose that in order to enable both expansion of infected cells and wart formation in vivo, MmuPV1 E8^E2 inhibits E4 protein expression in the basal keratinocytes that would otherwise undergo E4-mediated cell cycle arrest. IMPORTANCE Human papillomaviruses (PVs) initiate productive replication, which is characterized by genome amplification and expression of E4 protein strictly within suprabasal, differentiated keratinocytes. Mus musculus PV1 mutants that disrupt splicing of the E8^E2 transcript or abolish the interaction of E8^E2 with cellular NCoR/SMRT-HDAC3 co-repressor complexes display increased gene expression in tissue culture but are unable to form warts in vivo. This confirms that the repressor activity of E8^E2 is required for tumor formation and genetically defines a conserved E8 interaction domain. E8^E2 prevents expression of E4 protein in basal-like, undifferentiated keratinocytes and thereby their arrest in G2 phase. Since binding of E8^E2 to NCoR/SMRT-HDAC3 co-repressor is required to enable expansion of infected cells in the basal layer and wart formation in vivo, this interaction represents a novel, conserved, and potentially druggable target.

Dual targeting of protein translation and nuclear protein export results in enhanced antimyeloma effects.

Selinexor (KPT-330) is a small molecule inhibitor of XPO1, which mediates the transport of tumor suppressor proteins, oncogene messenger RNAs, and other proteins involved in governing cell growthfrom the cell nucleus to the cytoplasm. It is overexpressed in many cancer types. Because eukaryotic translation initiator factor 4E (eIF4E) plays a critical role in protein translation in cancer cells in multiple myeloma (MM), we evaluated the effectiveness of combined inhibition of protein translation and nuclear export in MM. Selinexor, an inhibitor of nuclear protein export, dose-dependently decreased eIF4E, IKZF1, and c-MYC protein levels. Using a doxycycline-inducible-pLKO-Tet-On vector, knockdown of eIF4E significantly enhanced the antiproliferative effects of selinexor, sensitized resistant MM cells to selinexor, and increased apoptosis in MM cells. Immunofluorescent analysis of MM cells showed that the combined treatment increased the localization of residual eIF4E to the nucleus compared with selinexor-only treatment. The overexpression of eIF4E at least partially rescued the effects of selinexor in MM cells by reducing G1 cell cycle arrest and increasing the selinexor-IC50 10-fold. Moreover, the combination of selinexor with pharmacologic inhibitors of protein translation showed synergistic anti-MM effects. These results suggest a synergistic anti-MM effect of selinexor combined with eIF4E inhibitors invitro. Our work provides a better understanding of the potential mechanism of resistance to selinexor and a rationale for combining selinexor with eIF4E inhibitors for the treatment of MM.

Intracellular Potassium in Cell Growth and Proliferation of Human Mesenchymal Stem Cells and Blood Lymphocytes

Many data show that K+ ions are essential for cell proliferation. In this brief review, we summarize our own studies and literature data that characterize the relationship between modulations of intracellular K+ content and the intensity of cell proliferation. Using flame emission assay we compared the transport of monovalent cations in transformed cells and in human mesenchymal stem cells in growing cultures, as well as during stress-induced cell cycle arrest and transition from monolayer (2D) to three-dimensional (3D) spheroids. A decrease in the intracellular content of K+ (evaluated as the ratio of the content of K+ in cells to the mass of cellular protein) associated with the accumulation of G1 cells in the population and accompanied by a stop in proliferation was revealed. The relationship between intracellular K+ and initiation of cell proliferation was further analyzed in human blood lymphocytes (HBL) as a model for the transition of cells from quiescence to proliferation. In HBL stimulated to proliferate, the content of K+ in cells increases during the transition G0/G1/S, accompanying the growth of small lymphocytes into blasts. Cellular water content, calculated from buoyant cell density, is higher in proliferating HBLs and in Jurkat leukemic T cells than in resting HBL. Available data suggest that intracellular K+ is important for successful cell proliferation as the main intracellular ion involved in the regulation of cell volume during the transition from quiescence to proliferation, and high K+ content and associated high water content in the cell are a characteristic feature of cell proliferation and transformation.

Guaiane‐type Sesquiterpenoid Dimers from Artemisia zhongdianensis and Antihepatoma Carcinoma Activity via the p38MAPK Pathway

Comprehensive Summary17 new guaiane‐type sesquiterpenoid dimers (GSDs), artemzhongdianolides B1—B17 (1—17), were isolated from Artemisia zhongdianensis under the guidance of bioassay, and elucidated by spectral analyses (HRESIMS, 1D and 2D NMR, IR, ECD). The absolute configuration of compounds 1, 3, 7, 9, 10, and 13 was determined by single‐crystal X‐ray diffraction analyses. Structurally, artemzhongdianolides B1 (1) and B2 (2) were the first example of the GSDs fused via a C‐13/C‐13' single bond, and artemzhongdianolides B3—B17 were [4 + 2] Diels–Alder adducts of two monomeric guaianolides. Most of the compounds showed antihepatoma cytotoxicity with IC50 values ranging from 9.9 to 170.1 μmol/L. Importantly, artemzhongdianolide B9 (9) was the most active one against three hepatoma cell lines with IC50 values of 13.1 μmol/L (HepG2), 19.5 μmol/L (Huh7), and 19.5 μmol/L (SK‐Hep‐1), and dose‐dependently inhibited cell migration and invasion, induced G1 cell cycle arrest and cell apoptosis in HepG2 cells. Compound 9 might suppress HepG2 cells via affecting the p38MAPK signaling pathway suggested by machine learning approach, and significantly upregulated expression of phosphorylated p38 validated by Western blot assay.

Investigation of Anticancer Properties of Monoterpene-Aminopyrimidine Hybrids on A2780 Ovarian Cancer Cells

The present study aimed to characterize the antiproliferative and antimetastatic properties of two recently synthesized monoterpene-aminopyrimidine hybrids (1 and 2) on A2780 ovary cancer cells. Both agents exerted a more pronounced cell growth inhibitory action than the reference agent cisplatin, as determined by the MTT assay. Tumor selectivity was assessed using non-cancerous fibroblast cells. Hybrids 1 and 2 induced changes in cell morphology and membrane integrity in A2780 cells, as evidenced by Hoechst 33258–propidium iodide fluorescent staining. Cell cycle analysis by flow cytometry revealed substantial changes in the distribution of A2780 ovarian cancer cells, with an increased rate in the subG1 and G2/M phases, at the expense of the G1 cell population. Moreover, the tested molecules accelerated tubulin polymerization in a cell-free in vitro system. The antimetastatic properties of both tested compounds were investigated by wound healing and Boyden chamber assays after 24 and 48 h of incubation. Treatment with 1 and 2 resulted in time- and concentration-dependent inhibition of migration and invasion of A2780 cancer cells. These results support that the tested agents may be worth of further investigation as promising anticancer drug candidates.

TACC3 is an independent prognostic marker, and knockdown of TACC3 enhances the efficacy of CDK1 inhibitor RO3306 in liver cancer cells.

The drug resistance of single-target therapy has gradually become an intractable clinical problem. Combination therapy may be an effective treatment to overcome or postpone drug resistance in cancer. Herein, we discussed the synergistic effect of transforming acidic coiled-coil containing protein 3 (TACC3) suppression and cyclin-dependent kinase 1 (CDK1) in hepatocellular carcinoma (HCC). The Cancer Genome Atlas database and bioinformatics methods were implemented to analyze the expression of CDK1 and TACC3, and predict the biological function of TACC3-related genes in HCC. In addition, in vitro experiments, including cell counting kit 8, transwell and flow cytometry were utilized to evaluate cell proliferation, migration, invasion, cell cycle arrest and apoptosis of HCC cells. Our results demonstrated that TACC3 is an unfavorable and independent prognostic factor to predict poor overall survival (OS) in HCC patients. Genetic inhibition of TACC3 exhibited a remarkable antineoplastic activity of HCC cell lines. Bioinformatic prediction proposed that CDK1 may be the main regulator of TACC3-related genes in HCC. In vitro experimental measurements suggested that a combination of si-TACC3 and CDK1 inhibitor synergistically inhibited cell proliferation and migration, and induced G2 cell cycle arrest and apoptosis of HepG2 or MHCC97H cells. In conclusion, our results revealed a prospective dual-target, TACC3 and CDK1, therapeutic strategy to improve the treatment of HCC.

Mechanisms of synergistic suppression of ALK-positive lung cancer cell growth by the combination of ALK and SHP2 inhibitors

Lung cancer is a major cause of cancer-related deaths. Alectinib is the first line of treatment for patients with ALK-positive lung cancer, but the survival rate beyond 2–3 years is low. Co-targeting secondary oncogenic drivers such as SHP2 is a potential strategy for improving drug efficacy. This is because SHP2 is expressed ubiquitously, but ALK expression is largely restricted to cancer cells. Thus, the combination of ALK and SHP2 inhibitors may provide a way to restrict synergistic cytotoxicity to cancer cells only, by reducing the dose of SHP2 inhibitors required for anticancer action and minimising SHP2-dependent systemic toxicity. The objective of this study was to investigate whether the combination of a SHP2 inhibitor (SHP099) with alectinib would synergistically suppress the growth of ALK-positive lung cancer cells. Our results demonstrated that the drug combination significantly and synergistically decreased cell viability at relatively low concentrations in ALK-positive H3122 and H2228 cells, due to G1 cell cycle arrest and increased apoptosis because of suppressed downstream RAS/MAPK signalling. The drug combination also induced the expression of mediators of the intrinsic apoptotic pathway, Bim and cleaved caspase-3, and modulated the expression of cell cycle mediators cyclin D1, cyclin B1, and phosphorylated CDK1.

PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.

#4208 CRP EXACERBATES SARS-COV-2-NUCLEOCAPSID PROTEIN-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims COVID-19, caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), is a progressive viral pneumonia with a broad spectrum of clinical manifestations involving multiple organs. Acute kidney injury (AKI) is common in COVID-19, especially the critically ill patients. Our previous study proved that kidney-specifically overexpressing SARS-CoV-2 N protein induces AKI, which become worsen in mice under ischemic condition. Clinically, CRP is a risk factor for AKI in COVID-19 patients. Recent clinical study showed that CRP Apheresis is able to effectively remove the plasma CRP and thus improve the clinical outcome in critically ill COVID-19 patients. We thus hypothesize that CRP may not only be a biomarker or risk factor, but also a potential mediator or novel therapeutic target for COVID-19 AKI. Method The role of CRP in COVID-19 AKI was investigated in a mouse model of AKI induced by ultrasound-microbubble-mediated kidney-specifically transferring SARS-CoV-2 N-expressing plasmids in CRP wide-type (WT) and Transgenic (Tg) mice. We also developed a novel therapy for SARS-CoV-2 N-induced by targeting CRP signaling with a neutralizing anti-CD32 antibody both in vivo and in vitro. Results In COVID-19 patients with AKI, serum levels of CRP were significantly elevated when compared with non-AKI group. In mice, kidney-specifically overexpressing SARS-CoV-2 N protein caused AKI including tubular necrosis and elevated levels of serum creatinine and BUN in CRP WT mice, which became more severe in CRP Tg mice. Importantly, targeting CRP-CD32 signaling with a neutralizing antibody to CD32 significantly inhibited SARS-CoV-2 N protein-induced AKI in CRP Tg mice. Mechanistically, we uncovered that CRP promoted SARS-CoV-2-N protein-induced AKI via the Smad3-p21-dependent G1 cell cycle arrest and NF-κB/p-p65-driven renal inflammation, which was blocked by anti-CD32 antibody in vivo and in vitro. Conclusion CRP is not only a biomarker or risk factor, but also a mediator in SARS-CoV-2 N protein-induced AKI. CRP signals through CD32 mediate SARS-CoV-2 N-induced AKI via mechanisms associated with the Smad3-p21-dependent G1 cell-cycle arrest and NF-κB/p-p65-driven renal inflammation. Targeting CRP-CD32 signaling may represent as a novel therapy for COVID-19-associated AKI.

Overexpression of long non-coding RNA GASL1 induces apoptosis and G0/G1 cell cycle arrest in human oral cancer cells

Oral cancer is one of the commonly reported malignancies of the human oral cavity and pharynx. It accounts for a significant level of cancer-based mortality across the globe. Long non-coding RNAs (lncRNAs) are emerging as important study targets in cancer therapy. The present study aimed to characterize the role of lncRNA GASL1 in regulating the growth, migration, and invasion of human oral cancer cells. The qRT-PCR showed significant (P&lt;0.05) upregulation of GASL1 in oral cancer cells. Overexpression of GASL1 led to the loss of viability of HN6 oral cancer cells by inducing apoptosis which was associated with upregulation of Bax and downregulation of Bcl-2. The apoptotic cell percentage increased from 2. 81% in control to 25.89% upon GASL1 overexpression. Cell cycle analysis showed that overexpression of GASL1 increased the G1 cells from 35.19% in control to 84.52% upon GASL1 overexpression indicative of G0/G1 cell cycle arrest. Cell cycle arrest was also accompanied by inhibition of cyclin D1 and CDK4 protein expression. Wound healing and transwell assays showed that overexpression of GASL1 significantly (P&lt;0.05) inhibited the migration and invasion of HN6 oral cancer cells. The invasion of the HN6 oral cancer cells was found to be decreased by more than 70%. Finally, the results of in vivo study revealed that GASL1 overexpression inhibits the xenografted tumor growth in vivo. Thus, the results are thus suggestive of the tumor-suppressive molecular role of GASL1 in oral cancer cells.

Serum-Stable Gold(III) Bisphosphine Complex Induces Mild Mitochondrial Uncoupling and In Vivo Antitumor Potency in Triple Negative Breast Cancer.

The preparation of cyclometalated complexes offers a path to stable materials, catalysts, and therapeutic agents. Here, we explore the anticancer potential of novel biphenyl organogold(III) cationic complexes supported by diverse bisphosphine ligands, Au-1-Au-5, toward aggressive glioblastoma and triple negative breast cancer cells (TNBCs). The [C^C] gold(III) complex, Au-3, exhibits significant tumor growth inhibition in a metastatic TNBC mouse model. Remarkably, Au-3 displays promising blood serum stability over a relevant therapeutic window of 24 h and alteration in the presence of excess L-GSH. The mechanism-of-action studies show that Au-3 induces mitochondrial uncoupling, membrane depolarization, and G1 cell cycle arrest and prompts apoptosis. To the best of our knowledge, Au-3 is the first biphenyl gold-phosphine complex to uncouple mitochondria and inhibit TNBC growth in vivo.

Prolonged cell cycle arrest by the CDK4/6 antagonist narazaciclib restores ibrutinib response in preclinical models of BTKi‐resistant mantle cell lymphoma

Introduction: 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 models. Methods: We compared the efficacy and safety of narazaciclib with the health authority approved CDKi in association with different BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We used the CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, and RNA sequencing followed by gene set enrichment analysis (GSEA), RT-PCR and western blot validations. In addition, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in an immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft. Results: Narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC50: 3.61 ± 2.1 µM), regardless of their sensitivity to ibrutinib. Although there was no correlation between CDKi sensitivity and activation 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 (NES > 2.5) upon narazaciclib treatment. This feature was associated 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 therapeutic acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTK-sensitive and BTK-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant (+10%–15%) augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome. Both the downregulation of phospho-histone H3 and the upregulation of p-p27/p27 and p16, also underwent a 10%–15% improvement in combination-treated cells. In vivo, while narazaciclib single agent achieved a 28% TGI in the CAM model, the narazaciclib-ibrutinib combination reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into the bone marrow, with no detectable toxicity. Conclusions: Narazaciclib, due to its completely distinct MoA from BTKi involving the direct modulation of the cell cycle, can achieve significant synergistic activity with ibrutinib in vitro and in vivo, especially in BTKi-resistant models of MCL. Ongoing phospho-proteomics and genetic edition assays will help deciphering the molecular bases of this unique drug cooperation at the cell cycle level. Encore Abstract - previously submitted to AACR 2023 The research was funded by: This study was financially supported by Onconova Therapeutics, Spanish Ministry of Science and Innovation (grant PID2021-123039OB-C21) (to GR). MFS holds a predoctoral fellowship (FI19/00338) from Instituto de Salud Carlos III. This work was carried out under the CERCA Program (Generalitat de Catalunya). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. A. Makovski-Silverstein Employment or leadership position: Onconova Therapeutics S. Cosenza Employment or leadership position: Onconova Therapeutics G. Roué Research funding: Onconova Therapeutics

Discovery, SAR and mechanistic studies of quinazolinone-based acetamide derivatives in experimental visceral leishmaniasis

Towards identification of novel therapeutic candidates, a series of quinazolinone-based acetamide derivatives were synthesized and assessed for their anti-leishmanial efficacy. Amongst synthesized derivatives, compounds F12, F27 and F30 demonstrated remarkable activity towards intracellular L. donovani amastigotes in vitro, with IC50 values of 5.76 ± 0.84 μM, 3.39 ± 0.85 μM and 8.26 ± 1.23 μM against promastigotes, and 6.02 μM ± 0.52, 3.55 ± 0.22 μM and 6.23 ± 0.13 μM against amastigotes, respectively. Oral administration of compounds F12 and F27 entailed >85% reduction in organ parasite burden in L. donovani-infected BALB/c mice and hamsters, by promoting host-protective Th1 cytokine response. In host J774 macrophages, mechanistic studies revealed inhibition of PI3K/Akt/CREB axis, resulting in a decrease of IL-10 versus IL-12 release upon F27 treatment. In silico docking studies conducted with lead compound, F27 demonstrated plausible inhibition of Leishmania prolyl-tRNA synthetase, which was validated via detection of decreased proline levels in parasites and induction of amino acid starvation, leading to G1 cell cycle arrest and autophagy-mediated programmed cell death of L. donovani promastigotes. Structure−activity analysis and study of pharmacokinetic and physicochemical parameters suggest oral availability and underscore F27 as a promising lead for anti-leishmanial drug development.

Neoadjuvant single-dose trilaciclib prior to combination chemotherapy in patients with early triple-negative breast cancer: Safety, efficacy, and immune correlate data from a phase 2 study.

603 Background: Trilaciclib, an intravenous CDK4/6 inhibitor, induces transient G1 cell cycle arrest. In preclinical studies, trilaciclib demonstrated immune-modulating effects by enhancing T-cell activation, favorably altering the tumor microenvironment (TME), and improving long-term immune surveillance. Preliminary data from this phase 2, single-arm, open-label study of neoadjuvant trilaciclib in triple-negative breast cancer (TNBC; NCT05112536) showed that single-dose trilaciclib increased CD8+ T-cell/Treg ratios within the TME. Here, we combine immune analysis with clinical outcomes to identify correlates of treatment response. Methods: Patients (pts) with early-stage TNBC received single-dose trilaciclib followed by trilaciclib + dose-dense anthracycline/cyclophosphamide and taxane (AC/T). Per investigator discretion, pts received pembrolizumab (pembro) 400 mg Q6W from cycle 1 and/or carboplatin (carbo) AUC 1.5 QW from cycle 5. Tumor biopsies and blood samples were collected prior to treatment, 7 days post single-dose trilaciclib, and during surgery, with an additional blood sample at cycle 2. PDL1 status was assessed per Ventana SP142 assay (positive immune cell score ≥ 1%). The primary objective is to evaluate the immune-based mechanism of action of single-dose trilaciclib. Results: As of January 3, 2023, 18/24 pts had completed treatment and/or had definitive surgery; 1 pt with neuroendocrine features discontinued due to disease progression. Median age was 57 years. At diagnosis, 79% of pts had stage II tumors (88% with ductal carcinoma; 38% with PDL1+ tumors). 18 pts received pembro + carbo, 3 pts pembro only, and 3 pts carbo only. Common treatment-related adverse events (TRAEs) were fatigue (79%), nausea, (67%), alopecia (67%), and neutropenia events (63%); 42% of pts had grade 3/4 neutropenia events. 4 serious TRAEs occurred in 2 (8%) pts: pembro-related colitis and hypertransaminasemia, trilaciclib/paclitaxel-related urosepsis, and AC-related febrile neutropenia. Response data were available for 15 pts; pathologic complete response (pCR) was observed in 7 (47%) pts, with pCR rates of 86% vs 13% for pts with PDL1+ vs PDL1– tumors, respectively. Immune analysis of the TME post trilaciclib monotherapy demonstrated several pCR-related features, such as increased stromal tumor-infiltrating lymphocytes and granzyme B+ cells. Correlative blood analysis revealed increased circulating baseline CD8+ T cells in pts achieving pCR. Conclusions: Safety and tolerability data are encouraging for trilaciclib in combination with AC/T ± pembrolizumab ± carboplatin in the neoadjuvant setting for early-stage TNBC. Preliminary efficacy data align with standard neoadjuvant chemotherapy regimens. Final pCR data from all pts and immune correlates from tumor and blood samples will be presented. Clinical trial information: NCT05112536 .

CDK4/6 nano-PROTAC enhances mitochondria-dependent photodynamic therapy and anti-tumor immunity

Cell cycle progression in cancer cells is highly activated, making CDK4/6 inhibition-based cell cycle arrest a potent cancer treatment strategy. To enhance therapeutic efficacy, it is crucial to explore CDK4/6 inhibition-based combination strategies. In this study, we found that combing CDK4/6 PROTAC with Chlorin e6-based photodynamic therapy produced a synergistic anti-cancer effect. This combination effect was mediated by mitochondria accumulation and activation, leading to increased production of reactive oxygen species and apoptosis. To promote clinical translation, we developed a self-assembled, carrier-free nanoparticle system to co-deliver these two molecules. The dual-drug nanoparticles not only induced higher apoptosis but also cooperatively induced immunogenic cell death and chemotaxis of immune cells, remodulating immunosuppressive tumor microenvironment to enhance anti-tumor immunity. This study provides a promising strategy to combine G1 cell cycle blockage and photodynamic therapy and advances the broad applications of PROTAC in clinical cancer treatment.

Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.

Immunomodulatory, apoptotic and anti-proliferative potentials of sildenafil in Ehrlich ascites carcinoma murine model: In vivo and in silico insights.

Sildenafil is a potent phosphodiesterase-5 (PDE5) inhibitor that effectively inhibits cGMP and increases the strength of nitric oxide. PDE5 was overexpressed in several carcinomas, including breast cancer, which inhibited tumor growth and cell division. The current research aims to investigate the in vivo sildenafil's immunomodulatory and antineoplastic potentials against Ehrlich Ascites Carcinoma. This study looked at the effects of sildenafil mono-treatment and co-treatment with cisplatin; tumor cell count, viability and the inhibition rate were determined. Apoptosis, cell cycle distribution, alterations in tumor cells and splenocytes proliferation, changes in splenocytes immunophenotyping using flowcytometry, plasma levels of malondialdehyde (MDA), reduced glutathione (GSH), interferone (IFN)-γ, granzyme B, alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine and hematological alterations were detected. Additionally, docking study was conducted to get further insights on how Sildenafil exerts its activity. Sildenafil mono-treatment and co-treatment with cisplatin markedly reduced tumor cell count, viability, growth rate and proliferative capability accompanied by apoptosis enhancement and G0/G1 and sub G1 cells cycle arrest. Fortunately, sildenafil evoked efficient cellular immune response by increasing plasma levels of granzyme B and IFN-γ, proportion of splenic T cytotoxic (CD3+CD8+) and T helper (CD3+CD4+), accompanied by decrease in the proportion of splenic regulatory T cells. . Moreover, in silico data suggest LcK and MAPKs as the potential targets of sildenafil. Furthermore, sildenafil rebalanced the oxidant-antioxidant status by decreasing MDA and increasing GSH plasma levels. Sildenafil successfully retrieved various hematological values besides renal and hepatic functions in EAC-bearing animals. In conclusion, our results suggest that sildenafil could be potential safe anti-tumor agent with immuno-modulatory properties against Ehrlich ascites carcinoma.