G0-G1 Arrest Research Articles

Comparative Study of Essential Oils from Tunisian Pinus Halepensis Mill. by Hydrodistillation and Microwave-Assisted Processes: Chemical Composition and Antioxidant and Cytotoxic Potential against Prostate and Cervical Cancer Cells.

Pinus halepensis Mill. is a Mediterranean aromatic plant largely used, in addition to its nutritional value, in traditional medicine as antiseptic, antifungal, antituberculotic, and antirheumatic. Thus, the objective of this work was to appraise the antioxidant and cytotoxic activity of the essential oil (EO) of P. halepensis from Tunisia on cancer cell cultures, along with chemical composition evaluation by GC-MS. To attain the best yield and also highest quality in extraction of the EOs, conventional hydrodistillation (HD) and novel microwave-assisted extraction (MAE) methods have been performed and compared. The antioxidant activity was evaluated through the inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH)• radicals. The cytotoxic activity in prostate cancer cells (LNCaP and PC3) and cervical cancer cells (HeLa) of EO was evaluated by the MTT assay and effect on the cell cycle by flow cytometry analysis. A total of 38 and 37 components were identified from HD (HD-EO)- and MAE (MAE-EO)-extracted EOs, respectively, which were dominated by hydrocarbon compounds (HD-EO = 86.65%; MAE-EO = 77.36%), especially monoterpenes (HD-EO = 32.11%; MAE-EO = 21.55%) and sesquiterpenes (HD-EO = 44.29%; MAE-EO = 61.32%). Both extracted EOs showed significant antioxidant activity, as shown by the inhibition of DPPH• radicals [IC50 (HD-EO) = 4102.30 ± 159.73 μg mL-1 and IC50 (MAE-EO) = 3430.13 ± 78.46 μg mL-1]. Also, the EOs exhibited substantial (p < 0.001) antiproliferative activities with G0-G1 arrest on PC3, LNCaP, and HeLa cells by yielding very low IC50 values more conspicuous in MAE-EO with respective IC50 values of 25.70 ± 6.58, 14.97 ± 3.21, and 14.55 ± 2.30 μg mL-1. This finding points out for the first time that the EO of P. halepensis Mill. from Tunisia can be an effective natural antitumor agent with more pronounced activity when extracted with the MAE method that, after further in vivo studies, can be harnessed as a putative phytopharmaceutical for prostate and cervical cancer treatment.

Design, synthesis, in vitro, and in silico studies of new thiadiazol derivatives as promising VEGFR-2 inhibitors and apoptosis inducers

The presented study reports the synthesis, in silico and in vitro evaluations of a series of novel thiadiazole derivatives designed with the pharmacophoric features of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) inhibitors. A comprehensive assessment against MCF-7 and HepG2 cancer cell lines revealed promising inhibitors, particularly, N-(4-((E)-1-(((Z)-5-acetyl-3-(2-chlorophenyl)-1,3,4-thiadiazol-2(3H)-ylidene) hydrazono) ethyl) phenyl)-4-chlorobenzamide (compound 22) exhibited a significantly low half-maximal inhibitory concentration (IC50) value against both MCF-7 and HepG2 cells of 0.03 µM and 0.39 µM, respectively. The rest of compounds exhibited also significant IC50 values against MCF-7 and HepG2 cells of (0.07–4.7 µM) and (0.12–8.75 µM), respectively. Furthermore, compound 22 displayed a VEGFR-2 inhibitory activity (VEGFR-2 = 0.042 µM) comparable to the established inhibitor sorafenib (VEGFR-2 = 0.041 µM). Detailed cell cycle analysis demonstrated that compound 22 induced a G0-G1 phase arrest of the MCF-7 cells (71.33 %), indicative of its potential to modulate cell cycle progression. Notably, compound 22 also exhibited a substantial increase in apoptosis in the MCF-7 cells, particularly in early (22.04 %) and late stages (13.94 %), underscoring its remarkable pro-apoptotic effect. Additionally, Compound 22 demonstrated a notable decrease in the migratory ability and wound healing capacity of MCF-7 cells. Computational studies, including molecular docking, revealed high binding affinities. Over a 200 ns MD simulation, the VEGFR-2-compound 22 complex initially displayed a rapid increase in RMSD followed by stability around 3.5 Å due to ligand conformational change while maintaining stable protein binding. This was confirmed by RMSF, SASA, RoG, and hydrogen bonding analysis. MM-GBSA, ProLIF, PCAT and FEL studies confirmed VEGFR-2-compound 22 complex binding at both dynamic and energetic levels. The computational ADMET studies demonstrated favorable pharmacokinetic properties. Furthermore, Density Functional Theory (DFT) studies confirmed the reactivity and stability of compound 22. These comprehensive findings underscore the potential of compound 22 as a lead candidate for further investigation in the development of VEGFR-2-targeted anticancer agents.

Aberrant ATM signaling and homology-directed DNA repair as a vulnerability of p53-mutant GBM to AZD1390-mediated radiosensitization.

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of TP53 mutation status in a panel of IDH1 wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs). AZD1390 suppressed radiation-induced ATM signaling, abrogated G0-G1 arrest, and promoted a proapoptotic response specifically in p53-mutant GBM in vitro. In a preclinical trial using 10 orthotopic GBM models, AZD1390/RT afforded benefit in a cohort of TP53-mutant tumors but not in TP53-WT PDXs. In mechanistic studies, increased endogenous DNA damage and constitutive ATM signaling were observed in TP53-mutant, but not in TP53-WT, PDXs. In plasmid-based reporter assays, GBM43 (TP53-mutant) showed elevated DNA repair capacity compared with that in GBM14 (p53-WT), whereas treatment with AZD1390 specifically suppressed homologous recombination (HR) efficiency, in part, by stalling RAD51 unloading. Furthermore, overexpression of a dominant-negative TP53 (p53DD) construct resulted in enhanced basal ATM signaling, HR activity, and AZD1390-mediated radiosensitization in GBM14. Analyzing RNA-seq data from TCGA showed up-regulation of HR pathway genes in TP53-mutant human GBM. Together, our results imply that increased basal ATM signaling and enhanced dependence on HR represent a unique susceptibility of TP53-mutant cells to ATM inhibitor-mediated radiosensitization.

Design, synthesis, and evaluation of VHL-based EZH2 degraders for breast cancer

EZH2 (enhancer of zeste homolog 2) is one of the most important histone methyltransferases (HMTs), and overexpression of EZH2 can lead to proliferation, migration and angiogenesis of tumor cells. But most of EZH2 inhibitors are only effective against some hematologic malignancies and have poor efficacy against solid tumors. Here, we report the design, synthesis, and evaluation of highly potent proteolysis targeting chimeric (PROTACs) small molecules targeting EZH2. We developed a potent and effective EZH2 degrader P4, which effectively induced EZH2 protein degradation and inhibited breast cancer cell growth. Further studies showed that P4 can significantly decrease the degree of H3K27me3 in MDA-MB-231 cell line, induce apoptosis and G0/G1 phase arrest in Pfeiffer and MDA-MB-231 cell lines. Therefore, P4 is a potential anticancer molecule for breast cancer treatment.

Baicalin suppress the development of polycystic ovary syndrome via regulating the miR-874-3p/FOXO3 and miR-144/FOXO1 axis

Context Polycystic ovary syndrome (PCOS) is a common and complex disease caused by endocrine and metabolic dysfunction in women of reproductive age. Baicalin is reported to ameliorate PCOS. Objective This study determines whether baicalin could affect the progression of PCOS. Materials and methods To establish an animal model of PCOS, female Sprague-Dawley (SD) rats were subcutaneously injected with dehydroepiandrosterone (DHEA, 60 mg/kg) for 20 days. Next, normal and PCOS mice were divided into 3 groups: control, PCOS, PCOS + Baicalin (20 mg/kg) groups. In addition, the levels of microRNA-874-3p (miR-874-3p) and microRNA-144 (miR-144) in ovarian tissues were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results Compared to the PCOS group, baicalin treatment significantly declined free testosterone (33.71 pg/mL vs. 56.05 pg/mL) and luteinizing hormone (LH; 3971.73 pg/mL vs. 5201.50 pg/mL) levels in rats with PCOS. Additionally, compared to the control group, 100 μM baicalin lessened miR-874-3p and miR-144 levels in human ovarian granulosa cells (KGN cells) by 36.87% and 32.57%, respectively. Furthermore, forkhead box O (FOXO) proteins FOXO1 and FOXO3 are the direct targets of miR-144 and miR-874-3p, respectively. Meanwhile, baicalin induced G0-G1 phase arrest (69.56 ± 3.7% at baicalin with 100 μM vs. 51.24 ± 3.2%, control) in KGN cells correlating with decreased p27 Kip1 (FOXO proteins downstream effector gene) expression by 55.5%; however, miR-874-3p or miR-144 overexpression could abolish this effect. Conclusions Baicalin could alleviate the symptoms of PCOS via regulating miR-874-3p/FOXO3 and miR-144/FOXO1 axis, demonstrating its potential utility in PCOS treatment.

Cancer History Avoids the Increase of Senescence Markers in Peripheral Cells of Amnestic Mild Cognitive Impaired Patients.

Epidemiological studies show that having a history of cancer protects from the development of Alzheimer's Disease (AD), and vice versa, AD protects from cancer. The mechanism of this mutual protection is unknown. We have reported that the peripheral blood mononuclear cells (PBMC) of amnestic cognitive impairment (aMCI) and Alzheimer's Disease (AD) patients have increased susceptibility to oxidative cell death compared to control subjects, and from the opposite standpoint a cancer history is associated with increased resistance to oxidative stress cell death in PBMCs, even in those subjects who have cancer history and aMCI (Ca + aMCI). Cellular senescence is a regulator of susceptibility to cell death and has been related to the pathophysiology of AD and cancer. Recently, we showed that cellular senescence markers can be tracked in PBMCs of aMCI patients, so we here investigated whether these senescence markers are dependent on having a history of cancer. Senescence-associated βeta-galactosidase (SA-β-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry; phosphorylated H2A histone family member X (γH2AX) by immunofluorescence; IL-6 and IL-8 mRNA by qPCR; and plasmatic levels by ELISA. Senescence markers that were elevated in PBMCs of aMCI patients, such as SA-β-Gal, Go-G1 arrested cells, IL-6 and IL-8 mRNA expression, and IL-8 plasmatic levels, were decreased in PBMCs of Ca + aMCI patients to levels similar to those of controls or of cancer survivors without cognitive impairment, suggesting that cancer in the past leaves a fingerprint that can be peripherally traceable in PBMC samples. These results support the hypothesis that the senescence process might be involved in the inverse association between cancer and AD.

SNHG18 inhibits bladder cancer cell proliferation by increasing p21 transcription through destabilizing c-Myc protein

BackgroundLong non-coding RNAs (lncRNAs) have been confirmed to play important roles in various cancers including bladder cancer (BC). The precise expression pattern of lncRNA small nucleolar RNA host gene 18 (SNHG18) in BC and its mechanisms of action have not been fully explored.Materials and methodsThe expression of SNHG18 was evaluated by RT-qPCR in bladder cancer clinical samples and human bladder cancer cell lines, and stable cell lines overexpressing SNHG18 were constructed. The effect of SNHG18 on the proliferation of bladder cancer cells was detected by soft agar colony formation test, ATP activity test and subcutaneous tumorigenesis model in nude mice. The specific mechanism of SNHG18 inhibition of bladder cancer proliferation was studied by flow cytometry, western blotting, dual luciferase reporter gene assay and protein degradation assay.ResultsWe found that SNHG18 is significantly downregulated in BC tissues and cell lines. Kaplan–Meier analysis showed that SNHG18 expression is positively correlated with survival in BC patients. Ectopic overexpression of SNHG18 significantly inhibited the proliferation of BC cells in vitro and in vivo. Further mechanistic investigations demonstrated that SNHG18 inhibited c-Myc expression by modulating the ubiquitination-proteasome pathway and that c-Myc is the critical transcription factor that mediates SNHG18 inhibition of BC growth by directly binding to the p21 promoter, which was attributed with significant p21 accumulation.ConclusionsSNHG18 promotes the transcription and expression of p21 by inhibiting c-Myc expression, leading to G0-G1 arrest and inhibiting the proliferation of bladder cancer cells. These findings highlight a novel cell cycle regulatory mechanism involving the SNHG18/c-Myc/p21 pathway in BC pathogenesis and could potentially lead to new lncRNA-based diagnostics and/or therapeutics for BC.

Rationale design and synthesis of new apoptotic thiadiazole derivatives targeting VEGFR-2: computational and in vitro studies.

This work presents the synthesis and in vitro, and in silico analyses of new thiadiazole derivatives that are designed to mimic the pharmacophoric characteristics of vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. A comprehensive evaluation of the inhibitory properties of the synthesized thiadiazole derivatives against the cancer cell lines MCF-7 and HepG2 identified several auspicious candidates. Among them, compound 14 showed remarkably low IC50 values of 0.04 μM and 0.18 μM against MCF-7 and HepG2, respectively. VEGFR-2 inhibitory evaluation of compound 14 revealed a promising IC50 value in the nanomolar range (103 nM). Further examination of the cell cycle revealed that compound 14 has the ability to stop the progression of the cell cycle in MCF-7 cells via G0-G1 phase arrest. Interestingly, compound 14 also demonstrated a noteworthy pro-apoptotic effect in MCF-7 cells, with notable increases in early apoptosis (16.53%) and late apoptosis (29.57%), along with a slight increase in the population of necrotic cells (5.95%). Furthermore, compound 14 showed a significant drop in MCF-7 cells' ability to migrate and heal wounds. Additionally, compound 14 promoted apoptosis by boosting BAX (6-fold) while lowering Bcl-2 (6.2-fold). The binding affinities of the synthesized candidates to their target (VEGFR-2) were confirmed by computational investigations, including molecular docking, principal component analysis of trajectories (PCAT), and molecular dynamics (MD) simulations. Additionally, compound 14's stability and reactivity were investigated using density functional theory (DFT). These thorough results highlight compound 14's potential as a lead contender for additional research in the creation of anticancer drugs that target VEGFR-2. This work establishes a foundation for promising thiadiazole derivatives for future therapeutic developments in anticancer- and angiogenesis-related scientific fields.

Chemical Constituents of the Deep-Sea-Derived Penicillium citreonigrum MCCC 3A00169 and Their Antiproliferative Effects.

Six new citreoviridins (citreoviridins J-O, 1-6) and twenty-two known compounds (7-28) were isolated from the deep-sea-derived Penicillium citreonigrum MCCC 3A00169. The structures of the new compounds were determined by spectroscopic methods, including the HRESIMS, NMR, ECD calculations, and dimolybdenum tetraacetate-induced CD (ICD) experiments. Citreoviridins J-O (1-6) are diastereomers of 6,7-epoxycitreoviridin with different chiral centers at C-2-C-7. Pyrenocine A (7), terrein (14), and citreoviridin (20) significantly induced apoptosis for HeLa cells with IC50 values of 5.4 μM, 11.3 μM, and 0.7 μM, respectively. To be specific, pyrenocine A could induce S phase arrest, while terrein and citreoviridin could obviously induce G0-G1 phase arrest. Citreoviridin could inhibit mTOR activity in HeLa cells.

Extrinsic apoptosis and senescence involved in growth kinetics of seminoma to cisplatin.

Seminoma is the most common type of testicular germ cell tumour and is highly sensitive to cisplatin therapy, which has not been fully elucidated. In this study, we comprehensively monitored dynamic changes of TCam-2 cells after cisplatin treatment. At an early stage, we found that both low and high concentrations of cisplatin induced the S-phase arrest in TCam-2 cells. By contrast, the G0G1 arrest was caused by cisplatin in teratoma NTERA-2 cells. Afterwards, high concentrations of cisplatin promoted the extrinsic apoptosis and high expressions of related genes (Fas/FasL-caspase-8/-3) in TCam-2 cells. However, when decreasing the cisplatin, the apoptotic cells were significantly reduced, and accompanied by cells showing senescence-like morphology, positive SA-β-gal staining and up-regulation of senescence-related genes (p53, p21 and p16). Furthermore, the cell cycle analysis revealed that most of the senescent TCam-2 cells were irreversibly arrested in the G2M phase. G2M arrest was also observed in NTERA-2 cells treated with a low concentration of cisplatin, while no senescence-related phenotype was discovered. In addition, we detected the γ-H2AX, a DNA damage marker protein, and reactive oxygen species (ROS) and found both of which were elevated with the increase of cisplatin in TCam-2 cells. In conclusion, the extrinsic apoptosis and senescence are involved in the growth kinetics of TCam-2 cells to cisplatin, which provide some implications for studies on cisplatin and seminoma.

Senescence Markers in Peripheral Blood Mononuclear Cells in Amnestic Mild Cognitive Impairment and Alzheimer's Disease.

Recent studies suggest that cellular senescence plays a role in Alzheimer’s Disease (AD) pathogenesis. We hypothesize that cellular senescence markers might be tracked in the peripheral tissues of AD patients. Senescence hallmarks, including altered metabolism, cell-cycle arrest, DNA damage response (DDR) and senescence secretory associated phenotype (SASP), were measured in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), amnestic mild cognitive impairment (aMCI) and AD patients. Senescence-associated βeta-galactosidase (SA-β-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry, while IL-6 and IL-8 mRNA were analyzed by qPCR, and phosphorylated H2A histone family member X (γH2AX) was analyzed by immunofluorescence. Senescent cells in the brain tissue were determined with lipofuscin staining. An increase in the number of senescent cells was observed in the frontal cortex and hippocampus of advanced AD patients. PBMCs of aMCI patients, but not in AD, showed increased SA-β-Gal compared with HCs. aMCI PBMCs also had increased IL-6 and IL8 mRNA expression and number of cells arrested at G0-G1, which were absent in AD. Instead, AD PBMCs had significantly increased p16 and p53 expression and decreased γH2Ax activity compared with HC. This study reports that several markers of cellular senescence can be measured in PBMCs of aMCI and AD patients.

Antitumor Effect of Glandora rosmarinifolia (Boraginaceae) Essential Oil through Inhibition of the Activity of the Topo II Enzyme in Acute Myeloid Leukemia.

It was previously shown that the antitumor and cytotoxic activity of the essential oil (EO) extracted from the aerial parts of Glandora rosmarinifolia appears to involve a pro-oxidant mechanism in hepatocellular carcinoma (HCC) and in triple-negative breast cancer (TNBC) cell lines. Its most abundant compound is a hydroxy-methyl-naphthoquinone isomer. Important pharmacological activities, such as antitumor, antibacterial, antifungal, antiviral and antiparasitic activities, are attributed to naphthoquinones, probably due to their pro-oxidant or electrophilic potential; for some naphthoquinones, a mechanism of action of topoisomerase inhibition has been reported, in which they appear to act both as catalytic inhibitors and as topoisomerase II poisons. Our aim was to evaluate the cytotoxic activity of the essential oil on an acute myeloid leukemia cell line HL-60 and on its multidrug-resistant (MDR) variant HL-60R and verify its ability to interfere with topoisomerase II activity. MTS assay showed that G. rosmarinifolia EO induced a decrease in tumor cell viability equivalent in the two cell lines; this antitumor effect could depend on the pro-oxidant activity of EO in both cell lines. Furthermore, G. rosmarinifolia EO reduced the activity of Topo II in the nuclear extracts of HL-60 and HL-60R cells, as inferred from the inability to convert the kinetoplast DNA into the decatenated form and then not inducing linear kDNA. Confirming this result, flow cytometric analysis proved that EO induced a G0-G1 phase arrest, with cell reduction in the S-phase. In addition, the combination of EO with etoposide showed a good potentiation effect in terms of cytotoxicity in both cell lines. Our results highlight the antitumor activity of EO in the HL-60 cell line and its MDR variant with a peculiar mechanism as a Topo II modulator. Unlike etoposide, EO does not cause stabilization of a covalent Topo II-DNA intermediate but acts as a catalytic inhibitor. These data make G. rosmarinifolia EO a potential anticancer drug candidate due to its cytotoxic action, which is not affected by multidrug resistance.

Abstract 5495: PHI-501, a potent and novel inhibitor of NRAS mutated acute myeloid leukemia

Abstract Purpose: Development of the first-in-class inhibitor of NRAS mutated acute myeloid leukemia. PHI-501 has potent activity against receptor tyrosine kinases and intracellular kinases related to STAT, AKT, and ERK signaling. Description: Preclinical testing of PHI-501 for efficacy against NRAS mutations both in vitro and in vivo xenografts. Summary: Mutations in the RAS genes (including KRAS, NRAS, and HRAS) are discovered in about 30% of all tumors. KRAS is the most frequently mutated gene in cancers found in pancreatic, colon, and lung, while NRAS mutations around the hot spots at codons 12, 13, and 61 are more common in AML (10.3%). PHI-501 is a novel small molecule inhibitor intended for the treatment of AML in patients expressing the N-RAS activating mutation. PHI-501 presented higher selectivity within a panel of 355 protein kinases. PHI-501 exerts their anti-proliferative effects by inducing apoptosis and G0-G1 arrest and significantly induce apoptotic markers (cleaved PARP and caspase 3) in both Ba/F3-NRAS-G12D and OCI-AML3 cells. The results of Western blot analysis showed that PHI-501 attenuate phosphorylation of p70S6K1, AKT, and p38 in OCI-AML3 After 21 days of the treatment in subcutaneous OCI-AML3 human AML xenograft model, PHI-501 showed significant inhibition of tumor growth (TGI) in a dose-dependent manner. Compared to the vehicle group, the 40 mg/kg group demonstrated a 74.0% lower average tumor volume (P&amp;lt;0.0001) and a 78.5% lower average tumor weight (P=0.0041). No significant body weight changes were observed in any of the groups. In toxicology and pharmacokinetic study, PHI-501 exhibited pharmacokinetic profiles favorable for oral administration in rodent, dog, and monkey and displayed acceptable in vivo and in vitro safety profiles. These results show the significant ability of PHI-501 to inhibit the progression of NRAS activated AML tumor growth, making it a strong candidate for the treatment of AML. Conclusion: The preclinical evaluation of PHI-501, a novel N-RAS inhibitor, showed clear evidence of anticancer activity for AML and improved efficacy in both in vitro and in vivo models. Consequently, PHI-501 is a new potent multi-kinase inhibitor with characteristics that warrant entry into human trials for the treatment of AML in patients expressing the N-RAS activating mutation. Citation Format: Ky-Youb Nam, Jung Hee Park, Kyung Ah Kim, Inje Shin, June H-J Han, Kyu-Tae Kim, Jeong Hyeok Yoon, Hanna Cho, Seung-Hye Choi, Sandip Sengupta, Taebo Sim. PHI-501, a potent and novel inhibitor of NRAS mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5495.

Reducing regorafenib toxicity by combining with dual JAK-HDAC inhibitor in colorectal cancer.

e15597 Background: In the US, colorectal cancer (CRC) is the third most common cancer. Patients receiving regorafenib, a multiple-kinase inhibitor, recommended to manage metastatic CRCs (mCRCs), has a modest improvement in median overall survival but it is associated with several toxicities. Our present study addresses regorafenib-induced toxicity concerns by combining regorafenib with a novel dual JAK-HDAC inhibitor (JAK-HDACi). The rationale for the dual inhibitor drug selection is due to the facts that the JAK/STAT/SOCS pathway is modulated in CRCs, and concurrent inhibition of JAK sensitizes solid tumors to HDACi. This study focused on evaluating the efficacy and reducing regorafenib-induced toxicity with this novel therapeutic combination in CRC preclinical models. Methods: We evaluated the toxicity of the JAK-HDACi, regorafenib, and their combination in normal colonic cells (CRL-1807) and their efficacy in CRC cell lines (HCT116, RKO, HT29, and SW480) exhibiting various statuses of p53, KRAS, BRAF, EGFR, and microsatellite instability, by conducting colony formation, cell proliferation, and cell cycle arrest assays. Kinome profiling and whole transcriptomic analysis were performed. Their efficacy was assessed in vivo in a CRC patient-derived xenograft (PDX) model, and experimental metastasis was evaluated in NSG mice using luciferase-tagged HT29 cells. Non-invasive, whole-body bioluminescence imaging was performed. Tumor tissues were harvested and stored at −80°C or prepared formalin-fixed paraffin-embedded blocks for Hematoxylin and Eosin (H&amp;E) and immunostaining. Serum analysis was performed to evaluate liver and kidney functions to assess the toxicity. Results: At 500 nM concentrations, there was no pronounced death of CRL-1807 cells, but reduced number of colonies in CRC cells. Drug treatments decreased phosphorylation of STAT3 and ERK1/2 and cell viability, wherein the reduction was robust in the combination. The combination reduced activity of various kinases, as evident through kinome profiling. In SW480 cells, the combination caused G0-G1 cell arrest and decreased the S phase. RNA-seq results revealed modulation of key pathways: apoptosis, ECM-receptor interaction, and focal adhesion. The PDX model showed that the combination treatment reduced tumor growth, as evidenced in H&amp;E staining with higher necrosis and reduced Ki67 staining. Experimental metastasis, bioluminescence imaging, and histological examination showed pronounced reduction in metastasis in mice treated with the combination. Serum chemistry profiles showed that the treatments did not cause systemic toxicity to mice used in either model. Conclusions: The combination therapy with the JAK-HDACi and regorafenib was more effective than the single agents with no evident toxicity. These findings lend credence to a clinical trial to assess this combination for treatment of patients with advanced CRC.

LncRNA RPSAP52 promotes cell proliferation and inhibits cell apoptosis via modulating miR-665/STAT3 in gastric cancer

ABSTRACT LncRNA RPSAP52 is a newly identified functional molecular in several cancers, but its role in gastric cancer (GC) is currently unclear. This study aimed to investigate the biofunction of lncRNA RPSAP52 in GC. Quantitative polymerase-chain reaction (RT-qPCR) was employed to analyze the gene level of lncRNA RPSAP52 and miR-665. Cell proliferation capacity was evaluated via CCK-8 and colony formation assay. Flow cytometry was applied to detect cell cycle and cell apoptosis. Hematoxylin-eosin staining was conducted for histopathological analysis. Immunochemical staining was carried out to detect expression level of ki-67. Subcellular fractionation was performed to explore the position of lncRNA RPSAP52. The binding relationship among lncRNA RPSAP52, miR-665 and STAT3 was verified via luciferase reporter assay. RNA pull down experiments were used to verify the binding relationship between lncRNA RPSAP52 and miR-665. The STAT3 level was evaluated via Western blot. LncRNA RPSAP52 is significantly elevated in GC cells. Deletion of lncRNA RPSAP52 restrained cell proliferation and induced G0-G1 phase arrest, while expediting apoptosis in GC cells. Tumor growth in vivo was suppressed following lncRNA RPSAP52 depletion. MiR-665 was verified as the target of lncRNA RPSAP52. A ceRNA-sponge mechanism of lncRNA RPSAP52 on miR-665 was identified. Meanwhile, miR-665 functions as STAT3 sponge. MiR-665 overexpression and STAT3 depletion served the same functions as lncRNA RPSAP52 depletion in GC cells. LncRNA RPSAP52 exerted anti-cancer effects via modulating miR-665/STAT3 in GC. Abbreviations: Gastric cancer (GC); Quantitative polymerase-chain reaction (RT-qPCR); Helicobacter pylori (H. pylori); Roswell Park Memorial Institute 1640 (RPMI 1640); fetal bovine serum (FBS); glyceraldheyde 3-phosphate dehydrogenase (GAPDH); propidium iodide (PI); Cell counting kit-8 (CCK-8); radioimmunoprecipitation assay (RIPA); sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); polyvinylidene fluoride (PVDF); enhanced chemiluminescence (ECL); Statistical Product and Service Solutions (SPSS); standard deviation (SD).

Constitutive β-Catenin Overexpression Represses Lncrna MIR100HG Transcription via HDAC6-Mediated Histone Modification in Colorectal Cancer.

It provides a novel downstream mechanism highlighting β-catenin action during colon carcinogenesis and may shed light for further therapeutic approaches.

Mir-4746 inhibits the proliferation of colorectal cancer cells in vitro and in vivo by targeting CCND1

Colorectal cancer (CRC) is a commonly seen malignant tumor manifesting itself in the digestive tract, but it remains unclear what is the molecular mechanism behind its occurrence and development, which can have a significant impact on the clinical diagnosis and treatment of CRC. According to some studies, microRNA (miRNA) plays an essential role in the occurrence and development of cancer. In spite of this, there are still many miRNAs that play an important role in the progression of CRC but have yet to be reported. In our research, it was found out that the expression of mir-4746 is significantly down-regulated in CRC tissues and cells, and that its expression level is closely associated with the tumor size and prognosis of clinical patients. As revealed by function and mechanism experiments, targeting CCND1 mRNA 3′-UTR, mir-4746 can promote the degradation of CCND1 mRNA, thus reducing the protein level of CCND1, leading to cell G0-G1 phase arrest, and ultimately inhibiting the proliferation of CRC cells. For the first time, our study reported the biological functions of mir-4746 and its preliminary mechanism of action, in addition to demonstrating that mir-4746 can be applied as both a potential prognostic marker and the therapeutic target for CRC.

Mesenchyme homeobox 2 has a cancer-inhibiting function in breast carcinoma via affection of the PI3K/AKT/mTOR and ERK1/2 pathways

A cancer-inhibiting role of mesenchyme homeobox 2 (MEOX2) has been observed in several malignancies. However, the association between MEOX2 and breast carcinoma has not been addressed. This research focused on investigating the possible relevance of MEOX2 in breast carcinoma. Initial expression analysis by TCGA data uncovered low levels of MEOX2 in breast carcinoma. We then confirmed that MEOX2 was poorly expressed in clinical tumor specimens of breast carcinoma by real-time quantitative PCR and immunoblotting assays. Moreover, low levels of MEOX2 in breast carcinoma patients were found to be correlated with reduced overall survival. A series of cellular function assays showed that the forced expression of MEOX2 had anticancer effects, including the inhibition of cell proliferation, the induction of G0-G1 phase arrest, the restraint of metastatic potential, and the enhancement of chemosensitivity. Further analysis revealed that MEOX2 negatively modulated the phosphatidyl-inositol-3 kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase (ERK1/2) pathways. Reactivation of AKT by a chemical activator reversed MEOX2-mediated anticancer effects. An in vivo xenograft assay validated the anticancer function of MEOX2 in breast carcinoma. Taken together, these data show that MEOX2 exerts a cancer-inhibiting role in breast carcinoma by affecting the PI3K/AKT/mTOR and ERK1/2 pathways. This work suggests MEOX2 as a new contributor for breast carcinoma progression, which may be a candidate target for anticancer therapy development.

Downregulation of microRNA-6125 promotes colorectal cancer growth through YTHDF2-dependent recognition of N6-methyladenosine-modified GSK3β.

BackgroundMicroRNAs (miRNAs), the key regulator of gene expression, and N6‐methyladenosine (m6A) RNA modification play a significant role in tumour progression. However, regulation of m6A‐modified mRNAs by miRNAs in colorectal cancer (CRC), and its effect on progression of CRC, remains to be investigated.MethodsExpression of miR‐6125 and YTH Domain‐Containing Family Protein 2 (YTHDF2) was detected by western blotting and immunohistochemistry. The effects of miR‐6125 and YTHDF2 on proliferative capacity of CRC cells were analysed using soft agar, ATP, CCK8 and EdU assays, and in animal experiments.ResultsMiR‐6125 expression was downregulated markedly in CRC, and expression correlated negatively with tumour size and prognosis. MiR‐6125 targeted the 3′‐UTR of YTHDF2 and downregulated the YTHDF2 protein, thereby increasing the stability of m6A‐modified glycogen synthase kinase 3 beta (GSK3β) mRNA. Increased GSK3β protein levels inhibited the expression of Wnt/β‐catenin/Cyclin D1 pathway‐related proteins, leading to G0‐G1 phase arrest and ultimately inhibiting the proliferation of CRC cells.ConclusionsMiR‐6125 regulates YTHDF2 and thus plays a critical role in regulating the Wnt/β‐catenin pathway, thereby affecting the growth of CRC. Collectively, these results suggest that miR‐6125 and YTHDF2 are potential targets for treatment of CRC.

Tadalafil enhances the therapeutic efficacy of BET inhibitors in hepatocellular carcinoma through activating Hippo pathway

Bromodomain and extraterminal (BET) proteins are promising therapeutic targets for hematological and solid tumors. However, BET inhibitor monotherapy did not show a significant therapeutic benefit for hepatocellular carcinoma (HCC) in preclinical trials. Here, we identified YAP/TAZ genes, as determinants for sensitivity to BET inhibitors. YAP/TAZ expression, especially TAZ, promote resistance to BET inhibitor. In addition, we analyzed that the mRNA level of PDE5 was positively correlated with YAP/TAZ based on TCGA database and demonstrated tadalafil, a PDE5 inhibitor, could block YAP/TAZ protein expression by activating Hippo pathway. Cotreatment with tadalafil and JQ-1 synergistically reduced YAP/TAZ protein expression, suppressed proliferation and induced G0-G1 arrest of cultured HCC cells. JQ-1 alone does not show significant benefits in a mouse model of HCC induced by c-Myc/N-Ras plasmids. In contrast, the combination, tadalafil and JQ-1, successfully suppressed tumor progression, enhanced antitumor immunity by improving the ratio of activated CD8 and extended the survival time of mice. Our data define the key role of YAP/TAZ in mediating resistance to BET inhibitor, described the PDE5/PKG/Hippo/YAP/TAZ axis and identified a common clinical drug that can be developed as an effective combined strategy to overcome BET inhibitor resistance in MYC/Ras-driven HCC.