Protein Levels Of CDK4 Research Articles

YKL-06-061 exerts antitumor effect through G1/S phase arrest by downregulating c-Myc and inhibition of metastasis via SIK1 upregulation in pancreatic cancer.

Pancreatic cancer ranks fourth among cancer-related deaths with a low 5-year overall survival rate of less than 13%. At present, treatment of pancreatic cancer is still based on chemotherapy, but the efficacy is limited. Thus, a novel therapeutic agent for pancreatic cancer therapy is urgently needed. A library of compounds was screened, and YKL-06-061, a selective inhibitor of salt-inducible kinases (SIKs), was discovered for its ability of inhibiting the proliferation and metastasis of pancreatic cancer cells in vitro and reducing the growth of xenografts in nude mice in vivo. The results from transcriptome analysis showed that YKL-06-061 influenced the mRNA levels of many genes related to c-Myc and SIK1 signals. Based on this, it was found that YKL-06-061 induced cell cycle arrest at the G1 phase and decreased the levels of c-Myc, CDK4, and cyclin D1 protein. At the same time, YKL-06-061 inhibited invasion and metastasis of cancer cells, increased the levels of SIK1 and E-cadherin protein, and lowered vimentin and ZEB-1. Moreover, YKL-06-061 effectively enhanced the antiproliferation of gemcitabine or doxorubicin in pancreatic cancer cells in a synergistic manner. Collectively, these findings implicate YKL-06-061 as a promising therapeutic agent for patients with pancreatic cancer.

Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells

As a member of peroxisome proliferator-activated receptor (PPAR) family, PPARG has been reported to be involved in glucolipid metabolism in various species. However, the function of PPARG in estradiol (E2) synthesis, apoptosis, and proliferation in goat ovarian granulosa cells (GCs) is unclear. In this study, we found that goat PPARG was expressed in GCs of all grades of follicles, and localized in the cytoplasm and nucleus of GCs. Transfection of small interfering RNA-PPARG2 (si-PPARG2) decreased E2 synthesis and the abundances of HSD3B and CYP19A1 mRNA and protein. It also promoted cell apoptosis with significant increases in the ratio of BAX/BCL-2 and Caspase3 mRNA and protein. Meanwhile, cell cycle was inhibited by si-PPARG2 transfection, accompanied by decreased mRNA levels of CDK4, CKD6, MYC, CCND1, CCND2, PCNA, and CCNB, increased mRNA level of P53, and decreased protein levels of CDK4, MYC, and CCND1. Furthermore, PPARG interference affected the mRNA and protein abundances of CREB as well as the phosphorylation of CREB but not PKA. In conclusion, our data suggest that PPARG plays an important role in regulating E2 synthesis, cell apoptosis, and proliferation of goat GCs, including the CREB expression and phosphorylation. These results provide evidences for the in-depth study of PPARG in the regulation of goat GCs function.

NaV1.1 contributes to the cell cycle of human mesenchymal stem cells by regulating AKT and CDK2.

Non-excitable cells express sodium voltage-gated channel alpha subunit 1 gene and protein (known as SCN1A and NaV1.1, respectively); however, the functions of NaV1.1 are unclear. In this study, we investigated the role of SCN1A and NaV1.1 in human mesenchymal stem cells (MSCs). We found that SCN1A was expressed in MSCs, and abundant expression of NaV1.1 was observed in the endoplasmic reticulum; however, this expression was not found to be related to Na+ currents. SCN1A-silencing reduced MSC proliferation and delayed the cell cycle in the S phase. SCN1A silencing also suppressed the protein levels of CDK2 and AKT (herein referring to total AKT), despite similar mRNA expression, and inhibited AKT phosphorylation in MSCs. A cycloheximide-chase assay showed that SCN1A-silencing induced CDK2 but not AKT protein degradation in MSCs. A proteolysis inhibition assay using epoxomicin, bafilomycin A1 and NH4Cl revealed that both the ubiquitin-proteasome system and the autophagy and endo-lysosome system were irrelevant to CDK2 and AKT protein reduction in SCN1A-silenced MSCs. The AKT inhibitor LY294002 did not affect the degradation and nuclear localization of CDK2 in MSCs. Likewise, the AKT activator SC79 did not attenuate the SCN1A-silencing effects on CDK2 in MSCs. These results suggest that NaV1.1 contributes to the cell cycle of MSCs by regulating the post-translational control of AKT and CDK2.

Effect of neratinib on HER2 mRNA stability via hsa-miR-23a-5p and efficacy of CDK4/6 inhibitors in HER2-low breast cancer.

e12550 Background: Traditional HER2-negative breast cancer sometimes includes HER2-low components, and CDK4/6 inhibitors are the first- and second-line treatments for advanced HER2-low breast cancer. Nonetheless, little is understood about how the stability of HER2 mRNA in HER2-low breast cancer affects the effectiveness of CDK4/6 inhibitor therapy and the associated intervention techniques. Methods: In order to investigate the impact of HER2-low on the effectiveness of CDK4/6 inhibitor in treating breast cancer, we first used HER2-0 and HER2-low breast cancer cells/mice as the study object and assessed the anti-proliferation effect of CDK4/6 inhibitor using CCK8 technique and monoclonal formation experiment. Second, we increased the treatment of anti-HER2 inhibitor neratinib and assessed its impact on the efficacy of HER2 low breast cancer CDK4/6 inhibitors. By reducing the expression of HER2 in HER2 low breast cancer cells, we analyzed and determined whether HER2 is the primary pathway that influences the poor efficacy of HER2 low breast cancer CDK4/6 inhibitors; At last, actinomycin D was used to detect the mRNA stability of HER2 in the treatment of HER2-low breast cancer by neratinib against CDK4/6 inhibitors, and then microRNA sequencing and luciferase reporter gene detection technology were used. To investigate the specific mechanism of action of neratinib on the mRNA stability of HER2 in the treatment of HER2-low breast cancer by CDK4/6 inhibitors. Results: Our study found that treatment with CDK4/6 inhibitors was significantly less effective against HER2-low breast cancer compared to HER2-0 subtype (P=0.005). Notably, in HER2-low subtype cells, HER2 knockdown significantly inhibited cell proliferation (P<0.001), G1 phase cell cycle arrest (P=0.001), and downregulated cyclin D1 expression and CDK4 protein levels (P=0.007, P=0.003). Second, it was found that in the cell and animal models of HER2-low breast cancer, the addition of the CDK4/6 inhibitor drug neratinib effectively reduced the stability of HER2 mRNA (P=0.001), inhibited the cell cycle of G1 phase (P=0.005). In addition, we observed that lowering the dose of neratinib to 1/2 (20 mg/kg) remained effective and well tolerated (P=0.002). Finally, we found that neratinib can reduce the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3'UTR region of HER2 (P<0.001), thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. Conclusions: We found that low expression of HER2 reduced the efficacy of CDK4/6 inhibitors in HER2-low breast cancer; Neratinib reduces the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3 'UTR region of HER2, thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. These findings provide a tolerable and effective two-drug combination regimen for patients with clinical HER2-low breast cancer.

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.

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.

Abstract P14: Efficacy of FHD-286 monotherapy and combinations against Menin inhibitor sensitive and resistant AML cells

Abstract BRG1 (SMARCA4) and BRM (SMARCA2) are the core ATPase within the BAF chromatin remodeler complexes. FHD-286 (Foghorn Therapeutics) is a potent, selective, oral inhibitor of BRG1/BRM in clinical development in AML. Present studies show that FHD-286 induces marked in vitro differentiation and loss of viability in AML cell lines and patient-derived (PD) AML cells with MLLr or mutant (mt) NPM1. FHD-286 treatment concordantly reduced genome-wide ATAC-Seq and RNA-Seq peaks, with negative enrichment at the gene-sets of MYC and E2F targets, mTORC1 signaling, translation initiation/elongation and DNA replication. FHD-286 reduced the occupancy of BRG1 and H3K27Ac on the chromatin. Mass spectrometry revealed FHD-286 reduced protein levels of c-Myc (and its targets), PLK1, BCL2, PU.1 and CDK4, but increased HEXIM1, CDKN1A/1B, CEBPα and CD11b levels. CyTOF analysis in the phenotypically characterized, PD, AML stem/progenitor cells with MLL1r or mtNPM1 showed decline in the protein levels of c-Myc, PU.1, BRG1, MEF2C, PBX3, BCL2 and MCL1, but increase in cleaved PARP and caspase-3 levels. In a PD xenograft (PDX) model of MLL1r AML, monotherapy with FHD-286 (oral gavage) for 4 to 6-weeks reduced AML burden and improved survival. FHD-286 treatment also significantly inhibited the AML-initiating potential, by reducing AML burden and improving survival of the re-engrafted mice with the FHD-286-treated PDX cells. Co-treatment with FHD-286 and BET inhibitor (OTX015), Menin inhibitor (MI, SNDX-50469), decitabine or venetoclax, exerted synergistic lethality in AML cell lines and PD AML cells with MLL1r or mtNPM1. Importantly, co-treatment with FHD-286 and OTX015, SNDX-5613 (oral gavage), venetoclax or decitabine vs each drug alone significantly reduced AML burden and improved survival in the PDX models of AML cells with MLLr or with mtNPM1, without significant toxicity. Notably, FHD-286 also induced significant lethality in MI-resistant AML cell lines and PD AML cells. Through repeated shocks with LD90 dosing of MI, followed by drug washout and recovery, we have generated MLL1r (MV4-11-MITR) and mtNPM1 (OCI-AML3-MITR) AML cell lines that are tolerant/resistant (TR) to MI (without exhibiting Menin mutations) with LD50 values greater than 1 µM. A domain-specific CRISPR screen in MV4-11-MITR cells identified several druggable co-dependencies (e.g., EP300 and MOZ) and co-enrichments (SMARCA4, CREBBP and BRD4) with MI. Accordingly, co-treatment with FHD-286 and OTX015, SNDX-50469 or CBP/p300 inhibitor GNE781 exerted synergistic lethality in MV4-11-MITR and OCI-AML3-MITR cells as well as in PD MITR AML cells with MLL1r or mtNPM1. FHD-286 and OTX015 or SNDX-5613 treatment also reduced AML burden in mice bearing OCI-AML3-MITR xenografts. These findings demonstrate pre-clinical efficacy of FHD-286-based rational combinations and underscore their promise against MI-sensitive or resistant AML with MLL1r or mtNPM1. Citation Format: Warren C Fiskus, Jessica Piel, Murphy Hentemann, Mike Collins, Christopher P Mill, Branko Cuglievan, Courtney D DiNardo, Kapil N Bhalla. Efficacy of FHD-286 monotherapy and combinations against Menin inhibitor sensitive and resistant AML cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P14.

Fatty acid binding protein 5 is a novel therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer and is one of the most common cancers with high mortality worldwide. Reprogrammed lipid metabolism plays crucial roles in HCC cancer cell survival, growth, and evolution. Emerging evidence suggests the importance of fatty acid binding proteins (FABPs) in contribution to cancer progression and metastasis; however, how these FABPs are dysregulated in cancer cells, especially in HCC, and the roles of FABPs in cancer progression have not been well defined. To understand the genetic alterations and expression of FABPs and their associated cancer hallmarks and oncogenes in contributing to cancer malignancies. We used The Cancer Genome Atlas datasets of pan cancer and liver hepatocellular carcinoma (LIHC) as well as patient cohorts with other cancer types in this study. We investigated genetic alterations of FABPs in various cancer types. mRNA expression was used to determine if FABPs are abnormally expressed in tumor tissues compared to non-tumor controls and to investigate whether their expression correlates with patient clinical outcome, enriched cancer hallmarks and oncogenes previously reported for patients with HCC. We determined the protein levels of FABP5 and its correlated genes in two HCC cell lines and assessed the potential of FABP5 inhibition in treating HCC cells. We discovered that a gene cluster including five FABP family members (FABP4, FABP5, FABP8, FABP9 and FABP12) is frequently co-amplified in cancer. Amplification, in fact, is the most common genetic alteration for FABPs, leading to overexpression of FABPs. FABP5 showed the greatest differential mRNA expression comparing tumor with non-tumor tissues. High FABP5 expression correlates well with worse patient outcomes (P < 0.05). FABP5 expression highly correlates with enrichment of G2M checkpoint (r = 0.33, P = 1.1e-10), TP53 signaling pathway (r = 0.22, P = 1.7e-5) and many genes in the gene sets such as CDK1 (r = 0.56, P = 0), CDK4 (r = 0.49, P = 0), and TP53 (r = 0.22, P = 1.6e-5). Furthermore, FABP5 also correlates well with two co-expressed oncogenes PLK1 and BIRC5 in pan cancer especially in LIHC patients (r = 0.58, P = 0; r = 0.58, P = 0; respectively). FABP5high Huh7 cells also expressed higher protein levels of p53, BIRC5, CDK1, CDK2, and CDK4 than FABP5low HepG2 cells. FABP5 inhibition more potently inhibited the tumor cell growth in Huh7 cells than in HepG2 cells. We discovered that FABP5 gene is frequently amplified in cancer, especially in HCC, leading to its significant elevated expression in HCC. Its high expression correlates well with worse patient outcome, enriched cancer hallmarks and oncogenes in HCC. FABP5 inhibition impaired the cell viability of FABP5high Huh7 cells. All these support that FABP5 is a novel therapeutic target for treating FABP5high HCC.

Mediator Complex Subunit 8:Expression in Gastric Cancer, Prognostic Significance,and Impact on Cell Cycle

Objective To investigate the expression and prognostic significance of mediator complex subunit 8 (MED8) in gastric cancer and its impact on the cell cycle.Methods The expression of MED8 in gastric cancer and adjacent tissues and its correlation with patients' prognosis were analyzed using public databases.A validation cohort of 104 patients who underwent radical resection for gastric cancer in the First Affiliated Hospital of Bengbu Medical College from June 2012 to July 2017 was included.The receiver operating characteristic curve was established to evaluate the predictive value of MED8 for postoperative 5-year survival.Bioinformatics tools were used to predict the biological roles of MED8 in gastric cancer.The effect of the MED8 level on the G1/S phase transition of gastric cancer cells (MGC-803) was analyzed via lentivirus transduction and flow cytometry.Western blotting was carried out to assess the impact of MED8 expression on the protein levels of cyclin-dependent kinase 4(Cdk4) and G1/S-specific cyclin-D1(CyclinD1) in MGC-803 cells.Results The high expression of MED8 in the gastric cancer tissue was associated with poor prognosis (P<0.001) and had prognostic significance (area under curve=0.733,P<0.001).Gene enrichment analysis suggested that MED8 may participate in the cell cycle process.Flow cytometry results revealed that the upregulation of MED8 expression promoted the transition of MGC-803 cells from the G1 phase to the S phase (P<0.001),while the downregulation of MED8 had the opposite effect (P<0.001).Western blotting showed increases in the protein levels of Cdk4 and CyclinD1 in MGC-803 cells with upregulated MED8 expression (all P<0.001),and decreases in the cells with downregulated MED8 expression (all P<0.001).Conclusion MED8 is highly expressed in gastric cancer and may affect its progression and prognosis by regulating the G1/S phase transition of gastric cancer cells.

IGF2BP2 Drives Cell Cycle Progression in Triple-Negative Breast Cancer by Recruiting EIF4A1 to Promote the m6A-Modified CDK6 Translation Initiation Process.

IGF2BP2 is a new identified N6-methyladenosine (m6A) reader and associated with poor prognosis in many tumors. However, its role and related mechanism in breast cancer, especially in triple-negative breast cancer (TNBC), remains unclear. In this study, it is found that IGF2BP2 is highly expressed in TNBC due to the lower methylation level in its promoter region. Functional and mechanical studies displayed that IGF2BP2 could promote TNBC proliferation and the G1/S phase transition of the cell cycle via directly regulating CDK6 in an m6A-dependent manner. Surprising, the regulation of protein levels of CDK6 by IGF2BP2 is related to the changes in translation rate during translation initiation, rather than mRNA stability. Moreover, EIF4A1 is found to be recruited by IGF2BP2 to promote the translation output of CDK6, providing new evidence for a regulatory role of IGF2BP2 between m6A methylation and translation. Downregulation of IGF2BP2 in TNBC cell could enhance the sensitivity to abemaciclib, a CDK4/6 inhibitor. To sum up, our study revealed IGF2BP2 could facilitate the translation output of CDK6 via recruiting EIF4A1 and also provided a potential therapeutic target for the diagnosis and treatment of TNBC, as well as a new strategy for broadening the drug indications for CDK4/6 inhibitors.

Porcupine quills keratin peptides induces G0/G1 cell cycle arrest and apoptosis via p53/p21 pathway and caspase cascade reaction in MCF-7 breast cancer cells.

Porcupine quills, a by-product of porcupine pork, are rich in keratin, which is an excellent source of bioactive peptides. The objective of this study was to investigate the underlying mechanism of anti-proliferation effect of porcupine quills keratin peptides (PQKPs) on MCF-7 cells. Results showed that PQKPs induced MCF-7 cells apoptosis by significantly decreasing the secretion level of anti-apoptosis protein Bcl-2 and increasing the secretion levels of pro-apoptosis proteins Bax, cytochrome c, caspase 9, caspase 3 and PARP. PQKPs also arrested the cell cycle at G0/G1 phase via remarkably reducing the protein levels of CDK4 and enhancing the protein levels of p53 and p21. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis identified nine peptides with molecular weights less than 1000 Da in PQKPs. Molecular docking results showed that TPGPPT and KGPAC identified from PQKPs could bind with p53 mutant and Bcl-2 protein by conventional hydrogen bonds, carbon hydrogen bonds and van der Waals force. Furthermore, the anti-proliferation impact of synthesized peptides (TPGPPT and KGPAC) was shown in MCF-7 cells. These findings indicated that PQKPs suppressed the proliferation of MCF-7 breast cancer cells by triggering apoptosis and G0/G1 cell cycle arrest. Moreover, the outcome of this study will bring fresh insights into the production and application of animal byproducts. © 2023 Society of Chemical Industry.

Andrographolide inhibits the proliferation and migration of vascular smooth muscle cells via PI3K/AKT signaling pathway and amino acid metabolism to prevent intimal hyperplasia

Andrographolide (AGP) exerts pharmacological effects when used for the treatment of cardiovascular disease, but the molecular mechanisms underlying its inhibitory effects on the proliferation and migration of vascular smooth muscle cells (VSMCs) and intimal hyperplasia (IH) are unknown. The proliferation and migration of VSMCs treated with AGP were examined using the CCK-8, flow cytometry, and wound healing assays. Expression levels of proteins related to cell proliferation and apoptosis were quantified. Multi-omics analysis with RNA-seq and metabolome was used to explore the potential molecular mechanism of AGP treatment. Additionally, an in vivo model was established through ligation of the left common carotid artery to identify the therapeutic potential of AGP in IH. Molecular docking and western blotting were performed to verify the mechanism discovered with multi-omics analysis. The results showed that AGP inhibited the proliferation and migration of cultured VSMCs in a dose-dependent manner and alleviated IH-related vascular stenosis. AGP significantly downregulated the protein levels of CDK1, CCND1, and BCL2 and upregulated the protein level of BAX. Gene expression profiles showed a total of 3,298 differentially expressed genes (DEGs) after AGP treatment, of which 1,709 DEGs had upregulated expression and 1,589 DEGs had downregulated expression. KEGG enrichment analysis highlighted the PI3K/AKT signaling pathway, verified with the detection of the activation of PI3K and AKT phosphorylation. Further GO enrichment combined with metabolomics analysis showed that AGP inhibition in cultured VSMCs involved the amino acid metabolic process, and the expression levels of the two key factors PRDM16 and EZH2, identified with PPI and docking analysis, were significantly inhibited by AGP treatment. In conclusion, our study showed that AGP inhibited VSMCs proliferation and migration by suppressing the PI3K/AKT signaling pathway and amino acid metabolism, which, in turn, improved IH.

Phase 1 Results of Bromodomain and Extraterminal Inhibitor PLX51107 in Combination with Azacitidine in Patients with Relapsed/Refractory Myeloid Malignancies.

Treatment outcomes in patients with relapsed/refractory (R/R) MDS and AML remains dismal. Based on both extensive pre-clinical data and emerging clinical data, treatment with bromodomain and extra-terminal domain inhibitors (BETi) is a potential approach for patients with high-risk myeloid malignancies. We conducted a phase I trial to study the safety and efficacy of PLX51107 (BETi) and azacitidine combination therapy in patients with R/R AML and high-risk (HR) MDS and studied mechanisms of resistance to the combination therapy. Thirty-seven patients with HR R/R MDS (n=4) and R/R AML (n=33) were treated. Sixteen patients (43%) had MECOM gene rearrangement and 7 other patients had TP53 mutation. Median prior number of therapies was three (range 1-9); 97% had received prior hypomethylating agent and 84% prior venetoclax. Overall response rate was 8/37 (22%): complete remission with incomplete platelet recovery (n=1); morphological leukemia-free state (n=2); hematologic improvement (n=5). The most common non-hematological toxicities were febrile neutropenia and pneumonia in 12 (32%) patients each; 6 patients (17%) had severe hyperbilirubinemia. RNA-Sequencing analysis of mononuclear cells harvested on treatment (day 3) vs pre-treatment showed significant changes in mRNA expressions in responders: downregulation of MYC, BCL2, IL7R and CDK6 and upregulation of HEXIM1, CD93, DCXR and CDKN1A. Immunoblot analyses confirmed reduction in protein levels of c-Myc, CDK6, BCL2 and BCL-xL, and induction of BRD4 and HEXIM1 protein levels in responders. In a heavily pre-treated patient cohort with R/R MDS and AML, PLX51107+ azacitidine was well-tolerated and resulted in modest clinical benefit.

Inhibition of Cell Proliferation and Cell Death by Apigetrin through Death Receptor-Mediated Pathway in Hepatocellular Cancer Cells.

Epidemiologic research recommends using flavonoids in the diet due to their overall health benefits. Apigetrin (Apigenin 7-O-glucoside) is a glycoside phytonutrient found in fruits and vegetables and known for different biological activities such as antioxidant and anti-inflammatory properties. Hepatocellular cancer (HCC) is a major health concern because of its adverse prognosis and side effects of chemotherapeutic agents. In the present study, we determine the impact of apigetrin on HepG2 cells and its cell death mechanism. Apigetrin reduced HepG2 cell proliferation with morphological changes and floating cells in treated cells. Colony formation and wound healing assays showed a reduced cell number in treatment groups. Further, we checked for the cell cycle through flow cytometry to understand the cell death mechanism. Apigetrin induced G2/M phase arrest in HepG2 cells by regulating Cyclin B1 and CDK1 protein levels in HepG2 cells. Annexin V and propidium iodide (PI) staining was performed to confirm the apoptotic cell population in treated groups. At the higher concentration, apigetrin showed a late apoptotic population in HepG2 cells. Chromatin condensation was also found in the treatment groups. Western blot analysis showed an increased expression of extrinsic apoptotic proteins such as FasL, Cleaved caspase 8, Cleaved caspase 3, and cleavage of PARP. In comparison, intrinsic apoptotic pathway markers showed no changes in Bax, Bcl-xL, and Cleaved caspase 9. Altogether, these findings strongly indicate that apigetrin causes cell death in HepG2 cells through the extrinsic apoptotic pathway, and that the intrinsic/mitochondrial pathway is not involved.

4-Terpineol attenuates pulmonary vascular remodeling via suppressing PI3K/Akt signaling pathway in hypoxia-induced pulmonary hypertension rats

The hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) plays a pivotal role in pulmonary arterial remodeling (PAR) of hypoxia-induced pulmonary hypertension (HPH). 4-Terpineol is a constituent of Myristic fragrant volatile oil in Santan Sumtang. Our previous study found that Myristic fragrant volatile oil alleviated PAR in HPH rats. However, the effect and pharmacological mechanism of 4-terpineol in HPH rats remain unexplored. Male Sprague-Dawley rats were exposed to hypobaric hypoxia chamber (simulated altitudes of 4500 m) for 4 weeks to establish an HPH model in this study. During this period, rats were intragastrically administrated with 4-terpineol or sildenafil. After that, hemodynamic indexes and histopathological changes were assessed. Moreover, a hypoxia-induced cellular proliferative model was established by exposing PASMCs to 3% O2. PASMCs were pretreated with 4-terpineol or LY294002 to explore whether 4-terpineol targeted PI3K/Akt signaling pathway. The PI3K/Akt-related proteins expression was also accessed in lung tissues of HPH rats. We found that 4-terpineol attenuated mPAP and PAR in HPH rats. Then, cellular experiments showed 4-terpineol inhibited hypoxia-induced PASMCs proliferation via down-regulating PI3K/Akt expression. Furthermore, 4-terpineol decreased the p-Akt, p-p38, and p-GSK-3β protein expression, as well as reduced the PCNA, CDK4, Bcl-2 and Cyclin D1 protein levels, while increasing levels of cleaved caspase 3, Bax, and p27kip1in lung tissues of HPH rats. Our results suggested that 4-terpineol mitigated PAR in HPH rats by inhibiting the proliferation and inducing apoptosis of PASMCs through suppression of the PI3K/Akt-related signaling pathway.

Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities.

The degradation tag (dTAG) system for target protein degradation can remove proteins from biological systems without the drawbacks of some genetic methods, such as slow kinetics, lack of reversibility, low specificity, and the inability to titrate dosage. These drawbacks can make it difficult to compare toxicity resulting from genetic and pharmacological interventions, especially in vivo. Because the dTAG system has not been studied extensively in vivo, we explored the use of this system to study the physiological sequalae resulting from CDK2 or CDK5 degradation in adult mice. Mice with homozygous knock-in of the dTAG sequence onto CDK2 and CDK5 were born at Mendelian ratios despite decreased CDK2 or CDK5 protein levels in comparison with wild-type mice. In bone marrow cells and duodenum organoids derived from these mice, treatment with the dTAG degrader dTAG-13 resulted in rapid and robust protein degradation but caused no appreciable change in viability or the transcriptome. Repeated delivery of dTAG-13 in vivo for toxicity studies proved challenging; we explored multiple formulations in an effort to maximize degradation while minimizing formulation-related toxicity. Degradation of CDK2 or CDK5 in all organs except the brain, where dTAG-13 likely did not cross the blood brain barrier, only caused microscopic changes in the testis of CDK2dTAG mice. These findings were corroborated with conditional CDK2 knockout in adult mice. Our results suggest that the dTAG system can provide robust protein degradation in vivo and that loss of CDK2 or CDK5 in adult mice causes no previously unknown phenotypes.

Effect of mir-92a-3p on hydroquinone induced changes in human lymphoblastoid cell cycle and apoptosis.

Hydroquinone (HQ), one of the metabolites of benzene in humans, has significant hepatotoxic properties. Chronic exposure to HQ can lead to leukemia. In a previous study by this group, we constructed a model of malignant transformation of human lymphoblastoid cells (TK6) induced by chronic exposure to HQ with significant subcutaneous tumorigenic capacity in nude mice. miR-92a-3p is a tumor factor whose role in HQ-induced malignant transformation is not yet clear. In the present study, raw signal analysis and dual-luciferase reporter gene results suggested that miR-92a-3p could target and regulate TOB1, and the expression level of miR-92a-3p was significantly upregulated in the long-term HQ-induced TK6 malignant transformation model, while the anti-proliferative factor TOB1 was significantly downregulated. To investigate the mechanism behind this, we inhibited miR-92a-3p in a malignant transformation model and found a decrease in cell viability, a decrease in MMP-9 protein levels, a G2/M phase block in the cell cycle, and an upregulation of the expression of G2/M phase-related proteins cyclinB1 and CDK1. Inhibition of miR-92a-3p in combination with si-TOB1 restored cell viability, inhibited cyclin B1 and CDK1 protein levels, and attenuated the G2/M phase block. Taken together, miR-92a-3p reduced the cell proliferation rate of HQ19 and caused cell cycle arrest by targeting TOB1, which in turn contributed to the altered malignant phenotype of the cells. This study suggests that miR-92a-3p is likely to be a biomarker for long-term HQ-induced malignant transformation of TK6 and could be a potential therapeutic target for leukemia caused by long-term exposure to HQ.

Integrated network pharmacology and experimental verification to investigate the mechanisms of YYFZBJS against colorectal cancer via CDK1/PI3K/Akt signaling.

Colorectal cancer (CRC) is a common digestive tract malignancy with rising incidence and morbidity worldwide during recent years. Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS), a traditional Chinese medicine formula, has showed positive effects against cancers. However, the mechanisms underlying its anticancer effects requires investigation. Information on bioactive compounds, potential YYFZBJS targets, and CRC-associated genes, was obtained from public databases. The key targets and ingredients as well their corresponding signaling pathways were identified using bioinformatic approaches, including Kyoto encyclopedia of genes and genomes (KEGG) analyses, gene ontology (GO), and protein-protein interaction (PPI). Subsequently, molecular docking was used to verify the main compounds-targets. Potential YYFZBJS therapeutic effects against CRC were validated in vitro and in vivo. Using pharmacological network analysis, 40 YYFZBJS active compounds and 21 potential anti-CRC targets were identified. YYFZBJS was an important regulator of CRC through various targets and signaling pathways, particularly the cell cycle and PI3K/AKT pathway. Additionally, YYFZBJS suppressed the proliferation of CRC cells. Flow cytometry showed that YYFZBJS induced apoptosis and cell cycle arrest in the G2/M phase. Western blotting analysis indicated that YYFZBJS reduced the protein levels of CDK1, p-AKT, and p-PI3K, without altering total PI3K and AKT protein levels. In vivo analysis found that YYFZBJS inhibited tumor growth and PI3K/AKT signaling in a mouse model of CRC. As predicted by network pharmacology and validated by the experimental results, YYFZBJS inhibited proliferation, induced apoptosis and arrested cell cycle progression in CRC by modulating the CDK1/PI3K/Akt signaling pathway.

Overexpressed Cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors.

CDK4/6 inhibitors are the standard treatment in advanced HR+/HER2- breast cancer patients. Nevertheless, the resistance to CDK4/6 inhibitors is inevitable and the strategies to overcome resistance are of great interest. Here, we show that the palbociclib-resistant breast cancer cells expressed significantly higher levels of Cyclin D1 and CDK4 proteins because of upregulated protein synthesis. Silencing Cyclin D1 or CDK4 led to cell cycle arrest while silencing Cyclin E1 or CDK2 restored the sensitivity to palbociclib. Furthermore, PI3K/mTOR pathway was hyper-activated in palbociclib-resistant cells, leading to more phosphorylated 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Targeting PI3K/mTOR pathway with a specific PI3Kα inhibitor (BYL719) or an mTOR inhibitor (everolimus) reduced the protein levels of Cyclin D1 and CDK4, and restored the sensitivity to palbociclib. The tumor samples expressed significantly higher levels of Cyclin D1, CDK4, p-AKT and p-4E-BP1 after progression on palbociclib treatment. In conclusion, our findings suggest that overexpressed Cyclin D1 and CDK4 proteins lead to the resistance to CDK4/6 inhibitor and PI3K/mTOR inhibitors are able to restore the sensitivity to CDK4/6 inhibitors, which provides the biomarker and rationale for the combinational use of CDK4/6 inhibitors and PI3K/mTOR inhibitors after CDK4/6 inhibitor resistance in breast cancer.

SPTBN2 Promotes the Progression of Thyroid Cancer by Accelerating G1/S Transition and Inhibiting Apoptosis.

Background Thyroid carcinoma (TC) is an increasingly common malignancy of endocrine organs, and its most frequently encountered histotype is papillary thyroid cancer (PTC). Identifying new potential gene alterations is important for completely elucidating the mechanism of PTC initiation and progression. Thus, we performed whole transcriptome sequence analysis (RNA-seq) on 79 PTC tissue samples and paired adjacent nontumor tissue samples to study the molecular mechanism of TC tumorigenesis and progression further. The results of RNA-seq analysis showed that spectrin beta, nonerythrocytic 2 (SPTBN2), was markedly overexpressed in PTC tissues relative to that in the paired nontumor tissues. Additionally, the analysis results for 502 PTC samples and 58 nontumor thyroid samples from The Cancer Genome Atlas dataset were consistent with our RNA-seq results. However, the molecular mechanisms and function of SPTBN2 in TC progression remain unknown. Methods We examined SPTBN2 gene expression in 48 papillary thyroid tumor tissues and paired adjacent normal thyroid tissues by using qRT-PCR. SPTBN2 expression in the TC cell lines was silenced by small interfering RNA. Then, the transfected TC cells were used to investigate the in vitro function of SPTBN2. Result The expression of SPTBN2 was significantly upregulated in our RNA-seq cohort, our local validated cohort, and TCGA RNA-seq cohort. The results of the in vitro experiment revealed that in TC cell lines, SPTBN2 downregulation considerably suppressed tumor cell proliferation, the cell cycle, migration, colony formation, and invasion and induced cell apoptosis. Furthermore, the protein levels of CCNE2, CDK2, CDK4, and Bcl-2 were downregulated, and those of P21, Bax, cleaved caspase-8, and cleaved caspase-3 had increased in transfected TC cells relative to in control TC cells. Conclusion The downregulation of SPTBN2 caused apoptosis and retarded G1/S cell cycle transition in TC cells. Thus, SPTBN2 may be a good candidate gene for TC diagnosis and therapy.