Cyclin CDK4 Research Articles

Effect of Spicatoside a on Anti-Osteosarcoma MG63 Cells through Reactive Oxygen Species Generation and the Inhibition of the PI3K-AKT-mTOR Pathway.

Osteosarcoma is a primary malignant tumor found in the bones of children and adolescents. Unfortunately, many patients do not respond well to treatment and succumb to the illness. Therefore, it is necessary to discover novel bioactive compounds to overcome therapeutic limitations. Liriope platyphylla Wang et Tang is a well-known herb used in oriental medicine. Studies have shown that metabolic diseases can be clinically treated using the roots of L. platyphylla. Recent studies have demonstrated the anticarcinoma potential of root extracts; however, the exact mechanism remains unclear. The aim of this study was to examine the anti-osteosarcoma activity of a single compound extracted from the dried roots of L. platyphylla. We purified Spicatoside A (SpiA) from the dried roots of L. platyphylla. SpiA significantly inhibited the proliferation of human osteosarcoma MG63 cells in a dose- and time-dependent manner. SpiA also regulated the expression of various downstream proteins that mediate apoptosis (PARP, Bcl-2, and Bax), cell growth (cyclin D1, Cdk4, and Cdk6), angiogenesis (VEGF), and metastasis (MMP13). The Proteome Profiler Human Phospho-Kinase Array Kit showed that the AKT signaling protein was a target of SpiA in osteosarcoma cells. We also found that SpiA suppressed the constitutive activation of the PI3K-AKT-mTOR-p70S6K1 signaling pathway. We further validated the effects of SpiA on the AKT signaling pathway. SpiA induced autophagosome formation and suppressed necroptosis (a form of programmed cell death). SpiA increased the generation of reactive oxygen species (ROS) and led to the loss of mitochondrial membrane potential. N-acetylcysteine (NAC)-induced inhibition of ROS generation reduced SpiA-induced AKT inhibition, apoptotic cell death, and anti-metastatic effects by suppressing cell migration and invasion. Overall, these results highlight the anti-osteosarcoma effect of SpiA by inhibiting the AKT signaling pathway through ROS generation, suggesting that SpiA may be a promising compound for the treatment of human osteosarcoma.

Binding Modalities and Phase-Specific Regulation of Cyclin/Cyclin-Dependent Kinase Complexes in the Cell Cycle.

Cyclin-dependent kinases (CDKs) are activated upon cyclin-binding to enable progression through the cell cycle. Dominant CDKs and cyclins in mammalian cells include CDK1, CDK2, CDK4, and CDK6 and corresponding cyclins A, B, D, and E. While only certain, "typical" cyclin/CDK complexes are primarily responsible for cell cycle progression, "atypical" cyclin/CDK complexes can form and sometimes perform the same roles as typical complexes. We asked what structural features of cyclins and CDKs favor the formation of typical complexes, a vital yet not fully explored question. We use computational docking and biophysical analyses to exhaustively evaluate the structure and stability of all CDK and cyclin complexes listed above. We find that binding of the complexes is generally stronger for typical than for atypical complexes, especially when the CDK is in an active conformation. Typical complexes have denser clusters, indicating that they have more defined cyclin-binding sites than atypical complexes. Our results help explain three notable features of cyclin/CDK function in the cell cycle: (i) why CDK4 and cyclin-D have exceptionally high specificity for each other; (ii) why both cyclin-A and cyclin-B strongly activate CDK1, whereas CDK2 is only strongly activated by cyclin-A; and (iii) why cyclin-E normally activates CDK2 but not CDK1. Overall, this work reveals the binding modalities of cyclin/CDK complexes, how the modalities lead to the preference for typical complexes versus atypical complexes, and how binding modalities differ between typical complexes. Our observations suggest targeting CDK catalytic actions through destabilizing their native differential cyclin interfaces.

Dietary secoisolariciresinol diglucoside crude extract improves growth through modulating rumen bacterial community and epithelial development in lambs.

Flaxseed lignans, types of polyphenolic compounds, primarily consist of secoisolariciresinol diglucoside (SDG). Natural plant extracts are becoming increasingly important as feed for ruminant animals. An underutilized plant bioactive component, SDG shows promising benefits for young ruminant production. The objective of this study was to assess the impact of SDG on rumen fermentation using an in vitro rumen simulation technology. Additionally, we tested the effects of SDG (0.20 g kg-1 body weight) on rumen development and production performance of lambs in a production setting. The in vitro addition of 100 mg L-1 SDG demonstrated significant regulatory effects, with a notable decrease in the acetate/propionate ratio (P < 0.05). Feeding trials revealed that SDG significantly increased average daily feed intake and average daily weight gain (P < 0.05), and reduced the acetate/propionate ratio (P < 0.05). This led to a significant increase in the relative abundance of Eubacterium ruminantium (P = 0.038) and Butyrivibrio (P = 0.002). Furthermore, it promoted rumen development and upregulated the relative expression of mRNA of Cyclin E1 and CDK2 in rumen epithelial cells (P < 0.05). The SDG extract optimizes the composition of rumen microbiota and the development of rumen epithelial cells, promoting the growth of pre-weaning lambs. The SDG additive exhibits potential as a novel growth promoter for ruminant animals, offering a promising solution for sustainable livestock production. © 2024 Society of Chemical Industry.

An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes.

More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile-cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

KIFC3 promotes the proliferation, migration and invasion of non-small cell lung cancer through the PI3K/AKT signaling pathway

KIFC3 is a member of the Kinesin superfamily proteins (KIFs). The role of KIFC3 in non-small cell lung cancer (NSCLC) is unknown. This study aimed to elucidate the function of KIFC3 in NSCLC and the underlying mechanism. Immunohistochemistry indicated that KIFC3 was highly expressed in NSCLC tissues and correlated with the degree of differentiation, tumor size, lymph node metastasis and TNM stage. MTT, colony formation and Transwell assays demonstrated that KIFC3 overexpression promoted the proliferation, migration and invasion of NSCLC cells in vitro, while KIFC3 knockdown led to the opposite results. The protein expression levels of PI3Kp85α and p-Akt were increased after KIFC3 overexpression, meanwhile the downstream protein expression levels such as cyclin D1, CDK4, CDK6, RhoA, RhoC and MMP2 were increased. This promotion effect could be inhibited by a specific inhibitor of the PI3K/Akt pathway, LY294002. Co-immunoprecipitation assays confirmed the interaction between endogenous/exogenous KIFC3 and PI3Kp85α. Tumor formation experiments in nude mice confirmed that KIFC3 overexpression promoted the proliferation, migration and invasion of NSCLC cells in vivo and performed its biological function through the PI3K/Akt signaling pathway.In conclusion, KIFC3 promotes the malignant behavior of NSCLC cells through the PI3K/Akt signaling pathway.

Differential effects of montelukast and zafirlukast on MDA‑MB‑231 triple‑negative breast cancer cells: Cell cycle regulation, apoptosis, autophagy, DNA damage and endoplasmic reticulum stress.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple‑negative breast cancer MDA‑MB‑231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription‑quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki‑67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti‑apoptotic protein Bcl‑2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3‑II and DNA damage markers, including cleaved PARP‑1, phosphorylated (p)‑ATM and p‑histone H2AX. The number of caspase 3/7‑positive cells was greater in montelukast‑treated cells compared with zafirlukast‑treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol‑requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast‑induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

Circular RNA hsa_circ_0000467 promotes colorectal cancer progression by promoting eIF4A3-mediated c-Myc translation

BackgroundColorectal cancer (CRC) is the second most common malignant tumor worldwide, and its incidence rate increases annually. Early diagnosis and treatment are crucial for improving the prognosis of patients with colorectal cancer. Circular RNAs are noncoding RNAs with a closed-loop structure that play a significant role in tumor development. However, the role of circular RNAs in CRC is poorly understood.MethodsThe circular RNA hsa_circ_0000467 was screened in CRC circRNA microarrays using a bioinformatics analysis, and the expression of hsa_circ_0000467 in CRC tissues was determined by in situ hybridization. The associations between the expression level of hsa_circ_0000467 and the clinical characteristics of CRC patients were evaluated. Then, the role of hsa_circ_0000467 in CRC growth and metastasis was assessed by CCK8 assay, EdU assay, plate colony formation assay, wound healing assay, and Transwell assay in vitro and in a mouse model of CRC in vivo. Proteomic analysis and western blotting were performed to investigate the effect of hsa_circ_0000467 on c-Myc signaling. Polysome profiling, RT‒qPCR and dual-luciferase reporter assays were performed to determine the effect of hsa_circ_0000467 on c-Myc translation. RNA pull-down, RNA immunoprecipitation (RIP) and immunofluorescence staining were performed to assess the effect of hsa_circ_0000467 on eIF4A3 distribution.ResultsIn this study, we found that the circular RNA hsa_circ_0000467 is highly expressed in colorectal cancer and is significantly correlated with poor prognosis in CRC patients. In vitro and in vivo experiments revealed that hsa_circ_0000467 promotes the growth and metastasis of colorectal cancer cells. Mechanistically, hsa_circ_0000467 binds eIF4A3 to suppress its nuclear translocation. In addition, it can also act as a scaffold molecule that binds eIF4A3 and c-Myc mRNA to form complexes in the cytoplasm, thereby promoting the translation of c-Myc. In turn, c-Myc upregulates its downstream targets, including the cell cycle-related factors cyclin D2 and CDK4 and the tight junction-related factor ZEB1, and downregulates E-cadherin, which ultimately promotes the growth and metastasis of CRC.ConclusionsOur findings revealed that hsa_circRNA_0000467 plays a role in the progression of CRC by promoting eIF4A3-mediated c-Myc translation. This study provides a theoretical basis and molecular target for the diagnosis and treatment of CRC.

Activation of lncRNA DANCR by H3K27 acetylation regulates proliferation of colorectal cancer cells

The long noncoding DANCR functions as a tumor oncogene in many cancers, including colorectal cancer (CRC). However, the molecular mechanism of DANCR in CRC has not been explored. This study probed the function and potential mechanism by which DANCR contributes to the progression of CRC. The obtained data indicated that DANCR is overexpressed in CRC tissues and cell lines. Knockdown of DANCR hindered CRC cell proliferation, which was mediated by cyclin D1 and CDK4. Bioinformatic analysis, luciferase reporter assays and subcellular fractionation verified that DANCR directly binds to miR-508-5p. Moreover, DANCR acts as a miR-508-5p ceRNA to regulate expression of ATF1. In addition, upregulation of DANCR is attributed to H3K27 acetylation at the promoter region. In conclusion, our study confirmed that activation of lncRNA DANCR by H3K27 acetylation has an oncogenic role in CRC progression and provides a potential therapeutic target for CRC.

Temporin-GHaK Exhibits Antineoplastic Activity against Human Lung Adenocarcinoma by Inhibiting the Wnt Signaling Pathway through miRNA-4516.

(1) Background: GHaK is derived from the antimicrobial peptide temporin-GHa by substituting the amino acid H with K to enhance its bactericidal activity. The present research aims to broaden the pharmacological potential of GHaK by exploring its antineoplastic activity against human lung adenocarcinoma. (2) Methods: The cell viability, migration, invasion, apoptosis, and cell cycle of A549 and PC-9 cells were tested after GHaK treatment. miRNA sequencing, RT-PCR, Western blotting, and luciferase reporter gene assay were further performed to reveal the potential mechanism. (3) Results: GHaK significantly suppressed cell viability, migration, and invasion; induced apoptosis; and caused cell cycle arrest in the G2/M and S phase in PC-9 and A549 cells, respectively. The miRNA sequencing results show a total of 161 up-regulated and 115 down-regulated miRNAs. Furthermore, the study identified six up-regulated miRNAs (miR-4516, miR-4284, miR-204-5p, miR-12136, miR-4463, and miR-1296-3p) and their inhibitory effects on the expressions of target genes (Wnt 8B, FZD2, DVL3, and FOSL1) caused by miR-4516 directly interacting with Wnt 8B. Western blotting revealed the down-regulation of p-GSK-3β, along with a decreased expressions of cyclin A1 and CDK2 in A549 cells and cyclin B1 and CDK1 in PC-9 cells. (4) Conclusions: Temporin-GHaK exhibits antineoplastic activity against human lung adenocarcinoma by inhibiting the Wnt signaling pathway through miRNA-4516.

Tris(1,3-dichloro-2-propyl) phosphate-induced cytotoxicity and its associated mechanisms in human A549 cells.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used organophosphorus flame retardant and has been detected in various environmental matrices including indoor dust. Inhalation of indoor dust is one of the most important pathways for human exposure to TDCIPP. However, its adverse effects on human lung cells and potential impacts on respiratory toxicity are largely unknown. In the current study, human non-small cell carcinoma (A549) cells were selected as a cell model, and the effects of TDCIPP on cell viability, cell cycle, cell apoptosis, and underlying molecular mechanisms were investigated. Our data indicated a concentration-dependent decrease in the cell viability of A549 cells after exposure to TDCIPP for 48h, with half lethal concentration (LC50) being 82.6µM. In addition, TDCIPP caused cell cycle arrest mainly in the G0/G1 phase by down-regulating the mRNA expression of cyclin D1, CDK4, and CDK6, while up-regulating the mRNA expression of p21 and p27. In addition, cell apoptosis was induced via altering the expression levels of Bcl-2, BAX, and BAK. Our study implies that TDCIPP may pose potential health risks to the human respiratory system and its toxicity should not be neglected.

Abstract 1157: Inhibition Of P90rsk Ameliorates Pdgf-bb-induced Vascular Smooth Muscle Cell Phenotypic Change And Neointimal Hyperplasia

Platelet-derived growth factor type BB (PDGF-BB) has been reported to induce vascular smooth muscle cell (VSMC) proliferation and migration, which are major events that are highly linked to vascular diseases such as atherosclerosis and restenosis. Although p90RSK has been shown to regulate multiple cellular processes including cell growth, proliferation, survival and motility through various downstream substrates in different cell types, the role of p90RSK on PDGF-BB-induced VSMC proliferation and migration, and vascular neointima formation remains unknown. In this study, we determine whether inhibition of p90RSK protects from PDGF-BB-induced cellular phenotypic change and investigate the molecular mechanisms underlying the effect of p90RSK inhibition. In cultured primary rat VSMCs, we found that pretreatment with FMK or BI-D1870, specific inhibitors of p90RSK, suppressed PDGF-BB-induced VSMC phenotypic alteration, ECM accumulation, proliferation, and migration. In addition, FMK and BI-D1870 repressed cell cycle regulatory genes cyclin D1 and Cdk4 induced by PDGF-BB, whereas inhibition of p90RSK augmented cyclin-dependent kinase inhibitor p27 downregulated by PDGF-BB in VSMCs, suggesting that p90RSK induces VSMC proliferation via cell cycle regulation of G0/G1 phase. Notably, using the mouse model of partial carotid ligation model, FMK was found to attenuate the neointimal hyperplasia of carotid arteries. These results suggest that p90RSK impedes PDGF-BB induced VSMC phenotypic switching, proliferation, migration, and neointima formation.

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

Abstract LB334: Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis

Abstract Introduction: Eicosanoid mediated mechanisms of hormone therapy resistance in ER+HER2- breast cancer are poorly understood. Cytochrome P450 arachidonic acid (AA) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to breast cancer progression by promoting mitochondrial oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to hormonal therapy resistance. In this study, the well-known letrozole resistant (LR) MCF-7 AC1 cell line overexpressing aromatase was selected for fulvestrant resistance (FR). A clonal cell line resistant to letrozole and fulvestrant (LR/FR) also exhibited resistance to palbociclib (PR) (MCF-7 AC1 LR/FR/PR). Xenograft tumors of this cell line were resistant to letrozole, fulvestrant, and palbociclib. Structure activity studies and modeling led to the development of CYP3A4 AA epoxygenase inhibiting biguanides hexyl cuban-1-yl biguanide (HCB) and fluorinated hexyl derivatives C5F2-HCB and C6F3-HCB. Fluorination on the hexyl moiety led to broader inhibition of EET regioisomer biosynthesis. These agents were tested for inhibition of proliferation, signaling, and OXPHOS/glycolysis balance in the MCF-7 AC1 LR/FR/PR cell line. Results: In the MCF-7 AC1 LR/FR/PR clonal cell line cyclin D1 and estrogen receptor (ER) expression were undetectable, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated 11, 40, 13, 10, and 12- fold (n=3, p&amp;lt;0.001) compared to the MCF-7 cell line. LC-MS analysis demonstrated 1.8, 1.4, and 1.2- fold higher total cellular levels of (±)8,9, (±)11,12, and (±)14,15-EET in the MCF-7 AC1 LR/FR/PR cell line compared to the MCF-7 cell line (n=3, p&amp;lt;0.05). C6F3-HCB suppressed cellular levels of EETs: (±)8,9-EET by 55% (n=3, p&amp;lt;0.031), (±)11,12-EET by 53% (n=3, p&amp;lt;0.029), and (±)14,15-EET by 60% (n=3, P&amp;lt;0.0001). The MCF-7 AC1 LR/FR/PR cell line was more potently inhibited by hexyl-cuban-1-yl biguanides compared to the MCF-7 cell line, exhibiting lower IC50 values (MCF-7 AC1 LR/FR/PR IC50 vs. MCF-7 IC50; HCB 4.1 vs. 7.0 uM; for C5F2-HCB 10 vs. 25 uM; C6F3-HCB: 5.4 vs. 12 uM; P values all &amp;lt;0.05). Measurement of spare respiratory capacity revealed that the MCF-7 AC1 LR/FR/PR cell line had 4.2% spare respiratory capacity compared to 34% for MCF-7, indicating a lower OXPHOS reserve for the resistant cells. Additionally, the MCF-7 AC1 LR/FR/PR cell line displayed a 2.4-fold higher extracellular acidification rate (ECAR) than the MCF-7 cell line, indicating a higher glycolysis rate (Warburg effect) in the resistant cells. Conclusion: Selection for fulvestrant resistance of MCF-7 AC1 cells resulted in upregulation of CYP3A4 and biosynthesis of hormone therapy resistance associated EETs while correlating with greater sensitivity to fluorinated hexyl-cuban-1-yl biguanides that inhibit EET biosynthesis. Citation Format: Zhijun Guo, Jianxun Lei, Allison Makovec, Swaathi Jayaraman, John R. Hawse, Carol Lange, Elizabeth Ambrose, Gunda I. Georg, Tony D'Assoro, Goetz P. Matthew, David A. Potter. Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB334.

Therapeutic Effects of Arctiin on Alzheimer's Disease-like Model in Rats by Reducing Oxidative Stress, Inflammasomes and Fibrosis.

Alzheimer's disease (AD) affects approximately 50 million people globally and is expected to triple by 2050. Arctiin is a lignan found in the Arctium lappa L. plant. Arctiin possesses anti-proliferative, antioxidative and anti-adipogenic. We aimed to explore the potential therapeutic effects of Arctiin on rats with AD by evaluating the expression of TLR4, NLRP3, STAT3, TGF-β, cyclin D1, and CDK2. AD was induced in rats by administering 70 mg/kg of aluminum chloride through intraperitoneal injection daily for six weeks. After inducing AD, some rats were treated with 25 mg/kg of Arctiin daily for three weeks through oral gavage. Furthermore, to examine the brain tissue structure, hippocampal sections were stained with hematoxylin/eosin and anti-TLR4 antibodies. The collected samples were analyzed for gene expression and protein levels of TLR4, NLRP3, STAT3, TGF-β, cyclin D1, and CDK2. In behavioral tests, rats showed a significant improvement in their behavior when treated with Arctiin. Microimages stained with hematoxylin/eosin showed that Arctiin helped to improve the structure and cohesion of the hippocampus, which was previously impaired by AD. Furthermore, Arctiin reduced the expression of TLR4, NLRP3, STAT3, TGF-β, cyclin D1, and CDK2. Arctiin can enhance rats' behavior and structure of the hippocampus in AD rats. This is achieved through its ability to reduce the expression of both TLR4 and NLRP3, hence inhibiting the inflammasome pathway. Furthermore, Arctiin can improve tissue fibrosis by regulating STAT3 and TGF-β. Lastly, it can block the cell cycle proteins cyclin D1 and CDK2.

Vitexicarpin Induces Apoptosis and Inhibits Metastatic Properties via the AKT-PRAS40 Pathway in Human Osteosarcoma.

Osteosarcoma, which has poor prognosis after metastasis, is the most common type of bone cancer in children and adolescents. Therefore, plant-derived bioactive compounds are being actively developed for cancer therapy. Artemisia apiacea Hance ex Walp. is a traditional medicinal plant native to Eastern Asia, including China, Japan, and Korea. Vitexicarpin (Vitex), derived from A. apiacea, has demonstrated analgesic, anti-inflammatory, antitumour, and immunoregulatory properties; however, there are no published studies on Vitex isolated from the aerial parts of A. apiacea. Thus, this study aimed to evaluate the antitumour activity of Vitex against human osteosarcoma cells. In the present study, Vitex (>99% purity) isolated from A. apiacea induced significant cell death in human osteosarcoma MG63 cells in a dose- and time-dependent manner; cell death was mediated by apoptosis, as evidenced by the appearance of cleaved-PARP, cleaved-caspase 3, anti-apoptotic proteins (Survivin and Bcl-2), pro-apoptotic proteins (Bax), and cell cycle-related proteins (Cyclin D1, Cdk4, and Cdk6). Additionally, a human phosphokinase array proteome profiler revealed that Vitex suppressed AKT-dependent downstream kinases. Further, Vitex reduced the phosphorylation of PRAS40, which is associated with autophagy and metastasis, induced autophagosome formation, and suppressed programmed cell death and necroptosis. Furthermore, Vitex induced antimetastatic activity by suppressing the migration and invasion of MMP13, which is the primary protease that degrades type I collagen for tumour-induced osteolysis in bone tissues and preferential metastasis sites. Taken together, our results suggest that Vitex is an attractive target for treating human osteosarcoma.

“Keep on ROCKIn”: Repurposed ROCK inhibitors to boost corneal endothelial regeneration

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

MiR-575/RIPK4 axis modulates cell cycle progression and proliferation by inactivating the Wnt/β-catenin signaling pathway through inhibiting RUNX1 in colon cancer.

Receptor interacting protein serine/threonine kinase 4 (RIPK4) is widely involved in human cancer development. Nevertheless, its role in colon cancer (COAD) has not been elucidated till now. Our research aimed at exploring the function and underlying molecular mechanism of RIPK4 in COAD progression. Through bioinformatic analyses and RT-qPCR, RIPK4 was discovered to be increased in COAD cells and tissues, and its high level predicted poor prognosis. Loss-of-function assays revealed that RIPK4 silencing suppressed COAD cell growth, induced cell cycle arrest, and enhanced cell apoptosis. In vivo experiments also proved that tumor growth was inhibited by silencing of RIPK4. Luciferase reporter assay validated that RIPK4 was targeted and negatively regulated by miR-575. Western blotting demonstrated that Wnt3a, phosphorylated (p)-GSK-3β, and cytoplasmic and nuclear β-catenin protein levels, β-catenin nuclear translocation, and Cyclin D1, CDK4, Cyclin E, and c-Myc protein levels were reduced by RIPK4 knockdown, which however was reversed by treatment with LiCl, the Wnt/β-catenin pathway activator. LiCl also offset the influence of RIPK4 knockdown on COAD cell growth, cell cycle process, and apoptosis. Finally, RIPK4 downregulation reduced RUNX1 level, which was upregulated in COAD and its high level predicted poor prognosis. RIPK4 is positively associated with RUNX1 in COAD. Overexpressing RUNX1 antagonized the suppression of RIPK4 knockdown on RUNX1, Wnt3a, p-GSK-3β, cytoplasmic β-catenin, nuclear β-catenin, Cyclin D1, CDK4, Cyclin E, and c-Myc levels. Collectively, miR-575/RIPK4 axis repressed COAD progression via inactivating the Wnt/β-catenin pathway through downregulating RUNX1.

The search for CDK4/6 inhibitor biomarkers has been hampered by inappropriate proliferation assays

CDK4/6 inhibitors are effective at treating advanced HR+ /HER2- breast cancer, however biomarkers that can predict response are urgently needed. We demonstrate here that previous large-scale screens designed to identify which tumour types or genotypes are most sensitive to CDK4/6 inhibitors have misrepresented the responsive cell lines because of a reliance on metabolic proliferation assays. CDK4/6-inhibited cells arrest in G1 but continue to grow in size, thereby producing more mitochondria. We show that this growth obscures the arrest using ATP-based proliferation assays but not if DNA-based assays are used instead. Furthermore, lymphoma lines, previously identified as the most sensitive, simply appear to respond the best using ATP-based assays because they fail to overgrow during the G1 arrest. Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib because it also restricts cellular overgrowth through off-target effects. DepMap analysis of screening data using reliable assay types, demonstrates that palbociclib-sensitive cell types are also sensitive to Cyclin D1, CDK4 and CDK6 knockout/knockdown, whereas the palbociclib-resistant lines are sensitive to Cyclin E1, CDK2 and SKP2 knockout/knockdown. Potential biomarkers of palbociclib-sensitive cells are increased expression of CCND1 and RB1, and reduced expression of CCNE1 and CDKN2A. Probing DepMap with similar data from metabolic assays fails to reveal these associations. Together, this demonstrates why CDK4/6 inhibitors, and any other anti-cancer drugs that arrest the cell cycle but permit continued cell growth, must now be re-screened against a wide-range of cell types using an appropriate proliferation assay. This would help to better inform clinical trials and to identify much needed biomarkers of response.

Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation

ObjectiveProtein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms. MethodsGC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression. ResultsThe expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin G1 regulation. ConclusionPDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.