Active RB causes visible changes in nuclear organization

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state.

Melatonin Induces Cell Cycle Arrest, Inhibits Cell Proliferation and Accelerates Apoptosis by Modulation of CDK4 in NSCLC

Purpose To explore whether melatonin affect the progression of cell cycle and exert anticancer activities via the modulation of CDK4 in NSCLC . Methods Cells treated with melatonin were used for assessing the anticancer effect of melatonin. Cells transfected with lentivirus for CDK4 upregulation or downregulation was constructed to evaluate the role of CDK4 in melatonin-induced anticancer effect. The protein and mRNA level of CDK4, PCNA and Bax were detected by western blotting and qRT-PCR. The application of flow cytometry was used for analyzing the distribution of cell cycle and apoptosis. Animal model of subcutaneous tumor was constructed and used for further study in vivo. Results We found that melatonin inhibited cell viability, colony formation, downregulated the expression of CDK4 and PCNA while upregulated the level of Bax. Besides, melatonin decreased the phosphorylation of ERK. Importantly, inhibition of ERK activation by PD98059 particapated in melatonin-induced downregulation of CDK4. Furthermore, melatonin led to G1 arrest and cell apoptosis. CDK4 knockdown enhanced melatonin-induced cell cycle arrest while CDK4 overexpression reversed the effect. Additionally, the animal experiment showed that melatonin decreased the level of CDK4 and inhibited tumor growth. However, the anti-tumor effect of melatonin was reversed by CDK4 overexpression. Conclusion Taken together, CDK4 involved in anti-cancer activities of melatonin. Melatonin led to G1 arrest, blocked G1-to-S transition, as a result, inhibited cell proliferation and accelerates apoptosis via suppressing CDK4 signaling. Targeting CDK4 inhibition and combining it with melatonin has protential to be a novel strategy for NSCLC.

Isolation and Identification of Andrographis paniculata (Chuanxinlian) and Its Biologically Active Constituents Inhibited Enterovirus 71-Induced Cell Apoptosis

Aim:Andrographis paniculata (Burm. f.) Nees (also known as Chuanxinlian in Chinese) of Acanthaceae family is one of the Chinese herbs reputed to be effective in the treatment of inflammation, infection, cold, and fever. Enterovirus 71 (EV71) is one of the most important enteroviruses that cause hand, foot, and mouth disease (HFMD) accompanied with neurological complication.Methods: To explore an anti-infective Chinese herb medicine, pure compounds isolated or synthesized analogues from A. paniculata (AP) ethyl acetate (EtOAc) extract are used to explore their anti-EV71-induced cytotoxicity. The antiviral activity was determined by cytopathic effect (CPE) reduction, and sub-G1 assays were used for measuring lysis and apoptosis of EV71-infected rhabdomyosarcoma (RD) cells. IFNγ-driven luciferase reporter assay was used to evaluate their potential roles in activation of immune responses.Results: Our data showed that EV71-induced sub-G1 phase of RD cells was dose dependently increased. Highly apoptotic EV71-infected RD cells were reduced by AP extract treatment. Ergosterol peroxide (4) has the most anti-apoptotic effect among these seven compounds. In addition, 3,19-O-acetyl-14-deoxy-11,12-didehydroandrographolide (8) synthesized from acetylation of compound 7 showed significantly better antiviral activity and the lowest sub-G1 phase of 6%–18%. Further investigation of IFNγ-inducer activity of these compounds showed that compounds 3, 6, 10, 11, and 12 had significantly higher IFNγ luciferase activities, suggesting their potential to promote IFNγ expression and thus activate immune responses for antivirus function.Conclusion: Our study demonstrated that bioactive compounds of AP and its derivatives either protecting EV71-infected RD cells from sub-G1 arrest or possessing IFNγ-inducer activity might be feasible for the development of anti-EV71 agents.

Altertoxin II, a Highly Effective and Specific Compound against Ewing Sarcoma

A screening program designed to identify natural products with selective cytotoxic effects against cell lines representing different types of pediatric solid tumors led to the identification of altertoxin II as a highly potent and selective cytotoxin against Ewing sarcoma cell lines. Altertoxin II, but not the related compounds altertoxin I and alteichin, was highly effective against every Ewing sarcoma cell line tested, with an average 25-fold selectivity for these cells as compared to cells representing other pediatric and adult cancers. Mechanism of action studies revealed that altertoxin II causes DNA double-strand breaks, a rapid DNA damage response, and cell cycle accumulation in the S phase. Our studies also demonstrate that the potent effects of altertoxin II are partially dependent on the progression through the cell cycle, because the G1 arrest initiated by a CDK4/6 inhibitor decreased antiproliferative potency more than 10 times. Importantly, the cell-type-selective DNA-damaging effects of altertoxin II in Ewing sarcoma cells occur independently of its ability to bind directly to DNA. Ultimately, we found that altertoxin II has a dose-dependent in vivo antitumor efficacy against a Ewing sarcoma xenograft, suggesting that it has potential as a therapeutic drug lead and will be useful to identify novel targets for Ewing-sarcoma-specific therapies.

Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth

To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis, and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

A selective CuII complex with 4-fluorophenoxyacetic acid hydrazide and phenanthroline displays DNA-cleaving and pro-apoptotic properties in cancer cells

The thin line between efficacy and toxicity has challenged cancer therapy. As copper is an essential micronutrient and is important to tumor biology, CuII complexes emerged as an alternative to chemotherapy; however, its biological properties need to be better understood. Thus, we report in vitro the antitumor effects of two CuII complexes named [Cu(4-fh)(phen)(ClO4)2] (complex 1) and [Cu(4-nh)(phen)(ClO4)2]·H2O (complex 2), in which 4-fh = 4-fluorophenoxyacetic acid hydrazide; 4-nh = 4-nitrobenzoic hydrazide and phen = 1,10-phenanthroline. Both complexes presented cytotoxic activity against tumor cells, but only complex 1 showed significant selectivity. Complex 1 also induced DNA-damage, led to G0/G1 arrest and triggered apoptosis, which was initiated by an autophagy dysfunction. The significant in vitro selectivity and the action mechanism of complex 1 are noteworthy and reveal this prodrug as promising for anticancer therapy.

Enhanced Anti-Cancer Potency Using A Combination of Oleanolic Acid and Maslinic Acid to Control Treatment Resistance in Breast Cancer.

The phosphatidylinositol 3-kinase/AKT/mammalian target of Rapamycin (PI3K/AKT/mTOR) pathway is a complex intracellular metabolic pathway leading to cell growth, and tumor proliferation plays an essential role in drug resistance in breast cancer. Therefore, in the present study, the anti-cancer effects of oleanolic acid (OA) and maslinic acid (MA) were investigated to improve the performance of the treatment strategy. We investigated the effect of OA and MA on cell viability using the WST-1 method. The synergistic effect of the combination was analyzed by isobologram analysis. In addition, the effects of the two compounds, individually and in combination, on apoptosis, autophagy, and the cell cycle were investigated in MCF7 cells. In addition, changes in the expression of PI3K/AKT/mTOR genes involved in apoptosis, cell cycle and metabolism were determined by quantitative RT-PCR. MA, OA, and a combination of both caused G0/G1 arrest. Apoptosis also increased in all treated groups. The autophagosomal LC3-II formation was induced 1.74-fold in the MA-treated group and 3.25-fold in the MA-OA-treated group. The combination treatment resulted in increased expression of genes such as GSK3B, PTEN, CDKN1B and FOXO3 and decreased expression of IGF1, PRKCB and AKT3 genes. The results showed that the combination of these two substances showed the highest synergistic effect at the lowest dose and using MA-OA caused cancer cells to undergo apoptosis. The use of combination drugs may reduce the resistance of cancer cells to treatment. However, further studies are needed to clarify cases.

Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2V617F cells

Background Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs. Methods Specimens of Jak2V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes. Results Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest. Conclusions Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.

Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.