S-phase Arrest Research Articles

Mechanisms of S-phase arrest and mitochondrial dysfunction in complex III by DHODH inhibitors in tumorigenic TNBC cells

Mechanisms of S-phase arrest and mitochondrial dysfunction in complex III by DHODH inhibitors in tumorigenic TNBC cells

EXTH-14. IRREVERSIBLE TARGETING OF THE C-MYC/TOPOISOMERASE I AXIS IN GLIOBLASTOMA: DUAL INHIBITION USING A NOVEL INDENOISOQUINOLINE INHIBITOR

Abstract Glioblastoma–the most common primary CNS malignancy–is notorious for its cellular and molecular complexity and uniform fatality. Overexpression of the c-MYC and TOPOISOMERASE 1 (TOP1) oncogenes is implicated in more than 50% of human cancers. They are involved in the pathogenesis of glioblastomas with overexpression correlating with decreased overall median survival in glioblastoma patients. LMP744 is a non-camptothecin indenoisoquinoline small molecule inhibitor designed to target the C-MYC/TOP1 axis. The compound is inherently stable, has high brain penetrance, and irreversibly binds to its target providing several advantages over the camptothecin class of drugs. Human glioblastoma cells treated with LMP744 show a dose-dependent decrease in viability with LD50 ranging between 50 and 100 nM compared to 100-200µM following treatment with temozolomide in the same cell lines. Treatment results in a pro-apoptotic effect in exposed cells as well as S-phase arrest. Protein arrays and confirmatory western blots demonstrate increased CHK2 and decreased p53 and GSK-a/ß in treated cells. Orthotopic xenotransplantation via stereotactic injection of human glioblastoma cells (7×105 cells/4 µL) in nude mice showed exquisite sensitivity to LMP744 without adverse events. Administration of 20 mg/kg of LMP744 via tail vein injection resulted in total eradication of tumor signal on bioluminescent imaging. These preliminary findings suggest that LMP744–and perhaps the class of non-camptothecin indenoisoquinolones–is a promising novel candidate for glioblastoma pharmacotherapy.

Phytochemical composition and anticancer effect of Akebia trifoliata seed in non-small cell lung cancer A549 cells

BackgroundAkebia trifoliata is a widely distributed medicine and food homology plant. Its fruit is used to treat tumors and the seed’s weight proportion exceeds 50%. However, the anti-tumor effect of A. trifoliata seed remains poorly studied. MethodsUHPLC-Q-Orbitrap-MS was used to identify the chemical components of A. trifoliata seed water extract (WE) and ethanolic extract (EE), and their cytotoxicity was evaluated using MTT assay. Further, colony formation and cell cycle assays examined the anti-proliferative effect of A. trifoliata seed EE. Next, morphology observation, AO/EB staining, Hoechst 33,258 staining, Annexin V-PE/7-AAD staining, and JC-1 assays were employed to detect apoptosis. The influence of A. trifoliata seed EE on A549 cell metastasis was assessed by wound healing and transwell invasion tests. In addition, western blotting and network pharmacology were applied to further analyze its anticancer mechanism. ResultsEighty-two components were characterized from the A. trifoliata seed WE and EE. For anticancer activity, A. trifoliata seed EE exhibited high cytotoxicity against cancer cells A549 (IC50: 52.38 ± 1.04 μg/mL) and NCI-H1299 (IC50: 70.29 ± 0.58 μg/mL) and revealed low cytotoxicity against non-cancer cells MRC-5 (IC50: 101.28 ± 2.84 μg/mL) and L929 (IC50: 113.44 ± 0.55 μg/mL). A. trifoliata seed EE suppressed A549 cell proliferation by inducing S-phase arrest via down-regulation of CDK2 and p21 as well as up-regulation of cyclin E1 and E2. It increased the Bax/Bcl-2 ratio, reduced mitochondrial membrane potential (ΔΨm), activated caspase-9 and caspase-3, cleaved PARP, and triggered apoptosis through the mitochondrial pathway. Besides, it repressed migratory and invasive ability by reducing MMP-2, MMP-9, and N-cadherin levels. ConclusionA. trifoliata seed EE exhibits outstanding anticancer properties and has the exploitation potential as an anticancer drug in the pharmaceutical industry.

Targeting ATR Kinase as a Strategy for Canine Lymphoma and Leukaemia Treatment.

Ataxia telangiectasia and Rad3-related (ATR) kinase is one of the main regulators of cell response to DNA damage and replication stress. Effectiveness of ATR targeting in human cancers has been confirmed in preclinical studies and ATR inhibitors are currently developed clinically in human oncology. In the presented study, we tested the anticancer efficacy of ATR inhibitor berzosertib in an in vitro model of canine haematopoietic cancers. Using MTT assay and flow cytometry, we assessed the cytotoxicity of berzosertib in four established canine lymphoma and leukaemia cell lines and compared it with its activity against noncancerous canine cells. Further, we estimated the level of apoptosis in berzosertib-treated cells via flow cytometry and assessed H2AX phosphorylation as a marker of DNA damage using western blot technique. In flow-cytometric analysis, we also evaluated potential synergism between berzosertib and chlorambucil and assessed the influence of berzosertib on cell cycle disturbances induced by the drug. The results demonstrated that berzosertib, even without additional DNA damaging agent, can be effective against canine lymphoma and leukaemia cells at concentrations that were harmless for noncancerous cells, although sensitivity of individual cancer cell lines varied greatly. Cell death occurred through caspase-dependent apoptosis via induction of DNA damage. Berzosertib also acted synergistically with chlorambucil, probably by preventing DNA damage repair as a consequence of S-phase arrest abrogation. In conclusion, ATR inhibition may provide a new therapeutic option for the treatment of canine lymphomas and leukaemias, but further studies are required to determine potential biomarkers of their susceptibility.

The Chinese Herbal Formula Weichang'an Reduces the Proliferation of Human Gastric Cancer Cells via Mitochondrial Apoptosis

Abstract Objective The aim of the study was to investigate the effects of Chinese herbal formula Weichang'an (WCA) on the proliferation and mitochondria-mediated apoptosis of human gastric cancer cells. Methods Cell Counting Kit-8 (CCK8) was used to evaluate the antiproliferative activity of WCA on MKN45 cells; Giemsa staining was used to investigate cell colony formation; flow cytometry was used to analyze cell cycle, apoptosis rate, and caspases activation; and Hoechst staining was used to analyze the morphology of cell nuclei. The mitochondrial membrane potential was analyzed by both flow cytometric measurements and fluorescence microscopy. Western blot was used to analyze the protein levels of pro-caspase-3, Bcl-2, Bax, and Bcl-X. MKN45 cells were subcutaneously injected into the right forelimb of 16 nude mice to establish the tumor xenograft model, and a total of 12 nude mouse tumor xenograft models were successfully created. The nude mice were divided into the control group and the WCA-treated group. The two groups received normal saline and WCA treatment at 35.49 g/kg by a single daily oral gavage for 21 days. The body weight and tumor size of the nude mice were measured twice a week. The ultrastructural changes of subcutaneous tumors were observed by a transmission electron microscope. Results WCA can suppress cell proliferation and colony formation. It can also induce changes in the mitochondrial membrane potential and increase the activities of caspases-3, caspases-8, and caspases-9 in MKN45 cells. WCA induced S-phase arrest and apoptosis in MKN45 cells, and decreased the expression levels of antiapoptotic proteins Bcl-2 and Bcl-X in MKN45 cells, while increasing the expression levels of the proapoptotic proteins Bax and pro-caspase-3 compared with the control group. WCA inhibited the growth of a xenografted MKN45 tumor in nude mice, and the protein levels of Bax and pro-caspase-3 were significantly increased, while Bcl-X and Bcl-2 were reduced compared with the control group. The differences are statistically significant (p < 0.05). Conclusion WCA could suppress the proliferation of gastric cancer cells via mitochondrial apoptosis.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

Design, synthesis and biological evaluation of novel naphthoquinothiazole derivatives as potent antitumor agents through inhibiting STAT3

The signal transducer and activator of transcription 3 (STAT3) has been established as a crucial drug target in the development of antitumor agents. In this study, a series of 21 derivatives of the STAT3 inhibitor napabucasin were designed and synthesized. Through preliminary screening against tumor cell lines, SZ6 emerged as the most potent compound with half maximal inhibitory concentration (IC50) values of 46.3 nM, 66.4 nM, and 53.8 nM against HCT116, HepG2, and Hela cells respectively. Furthermore, SZ6 effectively suppressed tumor invasion and migration in HCT116 cell assays by inducing S-phase arrest and apoptosis through inhibition of Protein Kinase B (PKB/AKT) activity and induction of reactive oxygen species (ROS). The mechanism underlying SZ6′s action involves inhibition of STAT3 phosphorylation, which was confirmed by western blotting analysis. Additionally, surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) demonstrated direct binding between SZ6 and STAT3. Notably, in vivo studies revealed that SZ6 significantly inhibited tumor growth without any observed organ toxicity. Collectively, these findings identify SZ6 as a promising STAT3 inhibitor for colorectal cancer treatment.

Green synthesis of zinc oxide nanoparticles using Rhus coriaria extract and their anticancer activity against triple-negative breast cancer cells

The growing interest in using plant extracts for the biogenic synthesis of zinc oxide nanoparticles (ZnO NPs) stems from their facile, eco-friendly, and biologically safe approach instead of chemical routes. For the first time, ZnO NPs were successfully biosynthesized using Rhus coriaria fruit aqueous extract as a reducing and capping agent. Characterization revealed that the biosynthesized ZnO NPs possessed a maximum absorbance of approximately 359 nm and closely resembled the hexagonal ZnO wurtzite crystalline structure, with an average crystalline size of 16.69 nm. The transmission electron microscope (TEM) showed the presence of spherical and hexagonal morphologies, with an average grain size of 20.51 ± 3.90 nm. Moreover, the elemental composition of the synthesized ZnO NPs was assessed via energy-dispersive X-ray spectrometry (EDX), and the presence of phytocompounds on their surface was subsequently verified through FT-IR analysis. The ζ-potential of ZnO NPs was recorded at − 19.9 ± 0.1663 mV. Regarding anti-cancer properties, ZnO NPs were found to possess potent anti-tumor effects on MCF-7 and MDA-MB-231 breast cancer cells. Their efficacy was dose-dependent, with IC50 values ranging from 35.04–44.86 μg/mL for MCF-7 and 55.54–63.71 µg/mL for MDA-MB-231 cells. Mechanistic studies in MDA-MB-231 cells revealed apoptosis induction, validated by DAPI staining, confocal microscopy, and Annexin V/PI staining, showing apoptosis by 12.59% and 81.57% at ½ IC50 and IC50 values, respectively. Additionally, ZnO NPs were observed to provoke S-phase arrest and inhibit colony-forming and metastatic potential by modulating apoptosis and metastasis-related genes. This study unravels new insights into how ZnO NPs provoke cancer cell death and inhibit metastasis, revealing new prospects in cancer nanotechnology.

Chondroitin sulfate-functionalized selenium nanoparticle-induced S-phase cell cycle arrest and apoptosis in HeLa Cells.

This study aimed to evaluate the anti-cervical cancer activity of chondroitin sulfate-functionalized selenium nanoparticles (SeCS) and to elucidate their action mechanism. Cytotoxic effect of SeCS on HeLa cells was assessed by MTT assay. Further molecular mechanism of SeCS was analyzed by flow cytometric assay and western blotting. The results showed that treatment with SeCS resulted in a dose- and time-dependent inhibition in the proliferation of HeLa cells. The data obtained from flow cytometry demonstrated that SeCS inhibited HeLa cell growth via the induction of S-phase arrest and cell apoptosis. Further mechanism analysis found that SeCS down-regulated expression levels of cyclin A and CDK2 and up-regulated p21 expression, which contributed to S arrest. Moreover, SeCS increased the level of Bax and decreased the expression of Bcl-2, resulting in the release of cytochrome C from mitochondria and activating caspase-3/8/9 for caspase-dependent apoptosis. Meanwhile, intracellular reactive oxygen species (ROS) levels were elevated after SeCS treatment, suggesting that ROS might be upstream of SeCS-induced S-phase arrest and cell apoptosis. These data show that SeCS has anti-tumor effects and possesses the potential to become a new therapeutic agent or adjuvant therapy for cancer patients. PRACTICAL APPLICATION: In our previous study, we used chondroitin sulfate to stabilize nano-selenium to obtain SeCS to improve the bioactivity and stability of nano-selenium. We found that it possessed an inhibitory effect on HeLa cells. However, the molecular mechanism remains unclear. This study elucidated the mechanism of SeCS damage to HeLa cells. SeCS has the potential to become a new therapeutic agent or adjuvant therapy for cancer patients.

Identification of RFC4 as a potential biomarker for pan-cancer involving prognosis, tumour immune microenvironment and drugs.

RFC4 is required for DNA polymerase δ and DNA polymerase ε to initiate DNA template expansion. Downregulated RFC4 inhibits tumour proliferation by causing S-phase arrest and inhibiting mitosis, resulting in the reduction of tumour cells. RFC4 has been implicated that it plays an important role in the initiation and progression of cancers, but a comprehensive analysis of the role of RFC4 in cancer has not been performed. We comprehensively analysed the expression, prognosis, methylation level, splicing level, relationship of RFC4 and immune infiltration, and pan-cancer immunotherapy response used various databases (including TCGA, GTEx, UALCAN, Oncosplicing, TIDE, TISCH, HPA and CAMOIP), and experimented its biological function in HCC. Through pan-cancer analysis, we found that RFC4 is significantly upregulated in most tumours. The tumour patients with high expression of RFC4 have poor prognosis. The methylation level and variable splicing level of RFC4 were abnormal in most tumours compared with the adjacent tissues. Furthermore, RFC4 was closely associated with immune cell infiltration in various cancers. RFC4 was significantly co-expressed with immune checkpoints and other immune-related genes. The expression of RFC4 could indicate the immunotherapy efficacy of some tumours. The RFC4 expression was associated with sensitivity to specific small molecule drugs. Cell experiments have shown that downregulated RFC4 can inhibit cell cycle and tumour cell proliferation. We conducted a systematic pan-cancer analysis of RFC4, and the results showed that RFC4 can serve as a biomarker for cancer diagnosis and prognosis. These findings open new perspectives for precision medicine.

Cymbopogon proximus phytochemicals induce S-phase arrest in A549 lung cancer cell lines via CDK2/cyclin A2 inhibition: gas chromatography-mass spectrometry and molecular docking analyses.

Cymbopogon proximus comprises several phytoconstituent classes that are reported to possess anticancer activity; however, studies on the anticancer potentials of theplant are lacking. C.proximus was extracted using solvents with increasing polarity. In-vitro cytotoxic activity of C.proximus extracts was examined against liver (HepG2), lung (A549), prostate (PC3), and bone (MG63) cell lines using MTT assay in comparison to doxorubicin. Flow cytometry was used to analyze the cell cycle for identification of the phase of inhibition. Chemical composition of the most active fraction was examined using the GC/MS technique. Molecular docking was used to explore the mechanism of cytotoxicity against A549, and the results were confirmed by Western blot analysis. Petroleum ether fraction was the highly effective fraction against A549 with IC50=14.02±2.79. GC/MS analysis of Pet.Eth led to the identification of nine compounds in unsaponifiable matter and 27 components in the saponifiable fraction. Di-N-octyl phthalate, 3-β-hydroxylean-11.13(18)-dien-30-oic acid methyl ester, elemol hydrocarbons, linoelaidic acid and linoleic acid demonstrated the lowest docking binding scores and similar binding modes against CDK2 as compared to that attained by the native ligand R-Roscovitine "CDK2 ATP inhibitor". Western blot analysis demonstrated that CDK2/cyclinA2 protein expression has been suppressed in A549 cell lines by Pet.Eth fraction.

Inhibition of the ATR-DNAPKcs-RB axis drives G1/S-phase transition and sensitizes triple-negative breast cancer (TNBC) to DNA holliday junctions

Targeting the DNA damage response (DDR) is a promising strategy in oncotherapy, as most tumor cells are sensitive to excess damage due to their repair defects. Ataxia telangiectasia mutated and RAD3-related protein (ATR) is a damage response signal transduction sensor, and its therapeutic potential in tumor cells needs to be precisely investigated. Herein, we identified a new axis that could be targeted by ATR inhibitors to decrease the DNA-dependent protein kinase catalytic subunit (DNAPKcs), downregulate the expression of the retinoblastoma (RB), and drive G1/S-phase transition. Four-way DNA Holliday junctions (FJs) assembled in this process could trigger S-phase arrest and induce lethal chromosome damage in RB-positive triple-negative breast cancer (TNBC) cells. Furthermore, these unrepaired junctions also exerted toxic effects to RB-deficient TNBC cells when the homologous recombination repair (HRR) was inhibited. This study proposes a precise strategy for treating TNBC by targeting the DDR and extends our understanding of ATR and HJ in tumor treatment.

Discovery of potent small molecule ubiquitin-specific protease 10 inhibitors with anti-hepatocellular carcinoma activity through regulating YAP expression

High expression of ubiquitin-specific protease 10 (USP10) promote the proliferation of hepatocellular carcinoma (HCC), thus the development of USP10 inhibitors holds promise as a novel therapeutic approach for HCC treatment. However, the development of selective USP10 inhibitor is still limited. In this study, we developed a novel USP10 inhibitor for investigating the feasibility of targeting USP10 for the treatment of HCC. Due to high USP10 inhibition potency and prominent selectivity, compound D1 bearing quinolin-4(1H)-one scaffold was identified as a lead compound. Subsequent research revealed that D1 significantly inhibits cell proliferation and clone formation in HCC cells. Mechanistic insights indicated that D1 targets the ubiquitin pathway, facilitating the degradation of YAP (Yes-associated protein), thereby triggering the downregulation of p53 and its downstream protein p21. Ultimately, this cascade leads to S-phase arrest in HCC cells, followed by cell apoptosis. Collectively, our findings highlight D1 as a promising starting point for USP10-positive HCC treatment, underscoring its potential as a vital tool for unraveling the functional intricacies of USP10.

Promoting the Anticancer Activity with Multidentate Furan-2-Carboxamide Functionalized Aroyl Thiourea Chelation in Binuclear Half-Sandwich Ruthenium(II) Complexes.

A new set of binuclear arene ruthenium complexes [Ru2(p-cymene)2(k4-N2OS)(L1-L3)Cl2] (Ru2L1-Ru2L3) encompassing furan-2-carboxamide-based aroylthiourea derivatives (H2L1-H2L3) was synthesized and characterized by various spectral and analytical techniques. Single-crystal XRD analysis unveils the N^O and N^S mixed monobasic bidentate coordination of the ligands constructing N, S, Cl/N, O, and Cl legged piano stool octahedral geometry. DFT analysis demonstrates the predilection for the formation of stable arene ruthenium complexes. In vitro antiproliferative activity of the complexes was examined against human cervical (HeLa), breast (MCF-7), and lung (A549) cancerous and noncancerous monkey kidney epithelial (Vero) cells. All the complexes are more efficacious against HeLa and MCF-7 cells with low inhibitory doses (3.86-11.02 μM). Specifically, Ru2L3 incorporating p-cymene and -OCH3 fragments exhibits high lipophilicity, significant cytotoxicity against cancer cells, and lower toxicity on noncancerous cells. Staining analysis indicates the apoptosis-associated cell morphological changes expressively in MCF-7 cells. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) analyses reveal that Ru2L3 can raise ROS levels, reduce MMP, and trigger mitochondrial dysfunction-mediated apoptosis. The catalytic oxidation of glutathione (GSH) to its disulfide form (GSSG) by the complexes may simultaneously increase the ROS levels, alluding to their observed cytotoxicity and apoptosis induction. Flow cytometry determined the quantitative classification of late apoptosis and S-phase arrest in MCF-7 and HeLa cells. Western blotting analysis confirmed that the complexes promote apoptosis by upregulating Caspase-3 and Caspase-9 and downregulating BCL-2. Molecular docking studies unfolded the strong binding affinities of the complexes with VEGFR2, an angiogenic signaling receptor, and BCL2, Cyclin D1, and HER2 proteins typically overexpressed on tumor cells.

Analysis of Vascular Smooth Muscle Cells from Thoracic Aortic Aneurysms Reveals DNA Damage and Cell Cycle Arrest as Hallmarks in Bicuspid Aortic Valve Patients.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.

MiR-380 inhibits the proliferation and invasion of cholangiocarcinoma cells by silencing LIS1

BackgroundThe objective of this study was to determine the role and regulatory mechanism of miR-380 in cholangiocarcinoma.MethodsThe TargetScan database and a dual-luciferase reporter assay system were used to determine if LIS1 was a target gene of miR-380. The Cell Counting Kit 8 assay, flow cytometry, and Transwell assay were used to detect the effects of miR-380 and LIS1 on the proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. Western blotting was used to determine the effect of miR-380 on MMP-2/p-AKT. Immunohistochemistry detected the regulatory effect of miR-380 on the expression of MMP-2/p-AKT/LIS1.ResultsExpression of miR-380 in cholangiocarcinoma was decreased but expression of LIS1 was increased. LIS1 was confirmed to be a target gene of miR-380. Transfection with miR-380 mimics inhibited the proliferation, S-phase arrest, and invasion of HCCC-9810/HuCCT1/QBC939 cells, and LIS1 reversed these inhibitory effects. miR-380 inhibitor promoted proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. si-LIS1 salvaged the promotive effect of miR-380 inhibitor. Overexpression of miR-380 inhibited expression of MMP-2/p-AKT/LIS1, but miR-380 inhibitor promoted their expression.ConclusionAn imbalance of miR-380 expression is closely related to cholangiocarcinoma, and overexpression of miR-380 inhibits the expression of MMP-2/p-AKT by directly targeting LIS1.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Abstract 1819: Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer

Abstract Background: Esculetin is a naturally occurring dihydroxycoumarin with anti-inflammatory, antioxidant, and anti-tumor activities. Copper ion transport proteins, notably SLC31A1 and ATP7B, are essential in maintaining intracellular copper homeostasis, averting disruptions that might lead to copper-induced cell death under normal physiological conditions. However, prior research has not explored Esculetin's role in inducing copper-induced cell death. Hence, this study aims to investigate the biological process of Esculetin-induced cell death and the molecular mechanism underlying dysregulation of intracellular copper ion homeostasis. Methods: Initially, the CCK-8 assay was used to determine the IC50 values of Esculetin on colorectal cancer cell lines (HCT116, HT29, SW480) and normal colonic epithelial cell lines (NCM460). In terms of biological behaviors, the effects of Esculetin on the proliferation of colorectal cancer cell lines were assessed using CCK-8, colony formation assay, and EdU staining. Flow cytometry was used to examine the impact of Esculetin on apoptosis and cell cycle distribution in colorectal cancer cells. A subcutaneous xenograft tumor model in nude mice was employed to evaluate the in vivo anti-tumor effects of Esculetin, while IHC staining was conducted to detect Ki-67 levels, and Western blotting was performed to assess EMT-related protein expression. To investigate copper ion homeostasis, qPCR was used to measure the expression levels of copper ion transporters SLC31A1 and ATP7B before and after Esculetin treatment. Results: The CCK-8 results revealed that the IC50 of Esculetin for normal colonic epithelial cell lines was significantly higher than that for colorectal cancer cell lines. Meanwhile, a notable inhibitory effect of Esculetin on the proliferation of colorectal cancer cells was observed. Additionally, Esculetin treatment suppressed colony formation and reduced the proportion of EdU-positive cells in colorectal cancer cells. Flow cytometry demonstrated an increase in apoptosis and S-phase arrest following Esculetin treatment. The subcutaneous xenograft tumor model showed significant inhibition of tumor growth by Esculetin, as evidenced by reduced Ki-67-positive cells in the Esculetin-treated group. Western blot analysis revealed a significant decrease in the expression levels of Vimentin and N-Cadherin and an increase in E-Cadherin expression. Furthermore, qPCR analysis post-Esculetin treatment showed an elevation in the inward copper transporter SLC31A1 levels, while the outward transporter ATP7B expression decreased. Conclusion: Esculetin induces dysregulation of intracellular copper ion homeostasis by upregulating SLC31A1 expression and downregulating ATP7B expression, leading to intracellular copper accumulation and subsequent disruption of copper homeostasis, thereby inducing copper-induced cell death. Citation Format: Fangyue Guo, Mengying Liu, Kaihe Zhu, Jiapeng Ru, Pei Li, Shanfeng Zhang. Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1819.

Abstract 510: Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer

Abstract The contemporary landscape of cancer treatment has witnessed significant progress with FDA-approved targeted drugs. The FDA's Project Optimus has underscored the growing need for innovative pharmacodynamic endpoints to determine the optimal biological dose in clinical development. Flow cytometry, a potent analytical tool in clinical and preclinical labs, now plays a leading role in cancer research, enabling the evaluation of new drug effects on cell cycle regulation and consideration of rational drug combinations with approved drugs. At TD2, we strive to enhance the translatability of in vitro induced cell cycle arrest studies to in vivo biomarker proof of concept and efficacy. We employ a comprehensive approach, utilizing flow cytometry to assess cell cycle dynamics in cultured cells and in vivo tumor models after treatment with approved and novel drugs. This abstract focuses on the translational relevance of combined in vitro and in vivo testing of well-known drugs that disrupt the cell cycle, laying the foundation for optimized treatment strategies and potentially personalized cancer therapies. Human A549 non-small cell lung carcinoma cells were treated with either thymidine (2.0mM) or the microtubule-depolymerizing drug nocodazole (100ng/ml) for 18 hours, then examined by flow cytometry. Thymidine induced a greater than 85% S-phase arrest, while nocodazole induced a greater than 88% G2/M-phase arrest. A549 cells (5 × 106 cells/mouse) were subcutaneously implanted in SCID mice, followed by treatment with an FDA-approved cell cycle inhibitor, Palbociclib, or cisplatin chemotherapy. Palbociclib, a CDK4/6 inhibitor, and low-dose cisplatin treatment resulted in an increased accumulation of cells in the G0/G1 phase, greater than 80% for each. Both treatments exhibited promising anti-cancer activity, with tumor growth inhibition (TGI) of 69% and 87%, respectively. Our findings highlight flow cytometry's multifaceted utility in assessing cell cycle arrest, providing a comprehensive view of agent effects on cancer cells in culture and within the complex in vivo tumor microenvironment. Determining the impact of any new drug on specific cell cycle checkpoints could allow for a more strategic plan for combination therapy. Integrating flow cytometry could enable early biomarker monitoring of tumor cell cycle changes, offering insights into the therapeutic potential of new agents alone or in combination. This multidimensional evaluation contributes to understanding cancer biology and has the potential to guide the development of novel therapeutic strategies, optimize treatment regimens to identify optimal biological doses, and improve patient outcomes in the era of precision medicine. Citation Format: Erin E. Trachet, Emily Cluff, Ana Lemos de Matos, Damaris Diaz, Chelsea Holmquist, Paul Gonzales. Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 510.

Abstract 4549: Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) represents a challenging subtype of breast cancer because of its limited treatment options. Recent research has highlighted the potential benefits of combining pharmacological agents to enhance therapeutic outcomes. Studies show that Ciclopirox olamine (CPX), an off-patent anti-fungal drug, induces cell cycle G1-phase arrest and activates the intrinsic apoptosis pathway by downregulating the anti-apoptotic proteins. Meanwhile, metformin, a safe and first-line drug for the treatment of type II diabetes, has been shown to induce cell cycle S-phase arrest and activates the extrinsic apoptosis pathway via induction of endogenous TRAIL in TNBC cells. However, whether the combinations of CPX and metformin would exert potent antitumor activity against TNBC remains unclear. Methods: Cell proliferation (MTS) assays and LIVE/DEAD cell imaging were used to determine the combined inhibitory effects on TNBC cells. IC50s and the combination index (CI) were calculated. Flow cytometry assays were carried out to analyze if the combinations are more potent to block cell cycle progression. Apoptotic-specific ELISA and flow cytometry analysis of Annexin V-PI staining were performed to assess if the combinations are significantly more effective to induce apoptosis. Western blot assays were used to examine if the combinations markedly induced the expression of TRAIL and cleavages of PARP and caspases. Tumor xenograft models will be established to test the in vivo antitumor activity of CPX and metformin either alone or in combinations. Results: Data indicated that both CPX and metformin independently inhibited the proliferation of TNBC cells. A synergistic effect was observed when CPX and metformin were used in combination, resulting in a more potent inhibition of TNBC cell proliferation. The combinatorial treatment effectively suppressed colony formation of TNBC cells. Synergy between CPX and metformin was partially linked to the enhanced activation of the AMPK-mTOR pathway, as the combination significantly enhanced the phosphorylation of AMPKα. The combinations also induced enhanced apoptosis in TNBC cells through both the intrinsic and extrinsic pathways. The combinatorial effects of CPX and metformin on in vivo tumor growth are under investigation. Conclusions: Combinations of CPX and metformin show synergistic effects on inhibiting TNBC cell proliferation. Our studies suggest a potential therapeutic strategy for TNBC, with the combinations of these agents exhibiting an enhanced antitumor activity. Further investigation is warranted to validate the results and explore their clinical applications. Key words: Triple-negative breast cancer, Ciclopirox olamine, Metformin, Combination, Apoptosis Citation Format: Yakun Wu, Hui Lyu, Congcong Tan, Margaret E. Larsen, Shile Huang, Bolin Liu. Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4549.