Discovery of Quinacrine as a Potent Topo II and Hsp90 Dual-Target Inhibitor, Repurposing for Cancer Therapy.

Topo II and Hsp90 are promising targets. In this study, we first verified the structural similarities between Topo IIα ATPase and Hsp90α N-ATPase. Subsequently, 720 compounds from the Food and Drug Administration (FDA) drug library and kinase library were screened using the malachite green phosphate combination with the Topo II-mediated DNA relaxation and MTT assays. Subsequently, the antimalarial drug quinacrine was found to be a potential dual-target inhibitor of Topo II and Hsp90. Mechanistic studies showed that quinacrine could specifically bind to the Topo IIα ATPase domain and inhibit the activity of Topo IIα ATPase without impacting DNA cleavage. Furthermore, our study revealed that quinacrine could bind Hsp90 N-ATPase and inhibit Hsp90 activity. Significantly, quinacrine has broad antiproliferation activity and remains sensitive to the multidrug-resistant cell line MCF-7/ADR and the atypical drug-resistant tumor cell line HL-60/MX2. Our study identified quinacrine as a potential dual-target inhibitor of Topo II and Hsp90, depending on the ATP-binding domain, positioning it as a hit compound for further structural modification.

CREB1 promotes proliferation and differentiation by mediating the transcription of CCNA2 and MYOG in bovine myoblasts.

The cAMP response element binding protein 1 (CREB1) is an important nuclear transcription factor in eukaryotes. To explore the potential role of CREB1 on Qinchuan bovine skeletal myoblasts, we investigated the function of CREB1 on proliferation and differentiation. In this study, we found that CREB1 promoted cell proliferation by promoting DNA synthesis in S phase and cell division in G2 phase and promoted myogenic differentiation process in bovine myoblasts. Through dual luciferase experiments, we found that CREB1 can bind to the proximal promoter regions of CCNA2 and MyoG, indicating that CREB1 can play a positive regulatory role in the proliferation and differentiation of myoblasts by mediating the transcription of CCNA2 and MyoG. In addition, through downstream target gene analysis and transcriptome sequencing, we found that CREB1 plays a role in cell proliferation, myogenic differentiation, skeletal muscle repair and other related pathways.

Genomic Variation-Mediating Fluconazole Resistance in Yeast.

Fungal infections pose a serious and growing threat to public health. These infections can be treated with antifungal drugs by killing hazardous fungi in the body. However, the resistance can develop over time when fungi are exposed to antifungal drugs by generating genomic variations, including mutation, aneuploidy, and loss of heterozygosity. The variations could reduce the binding affinity of a drug to its target or block the pathway through which drugs exert their activity. Here, we review genomic variation-mediating fluconazole resistance in the yeast Candida, with the hope of highlighting the functional consequences of genomic variations for the antifungal resistance.

Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells.

The accuracy of sixteen commonly used internal reference genes was assessed in skeletal muscle-derived satellite cells of Qinchuan cattle at different stages of proliferation and induction of differentiation to determine the most suitable ones. Quantitative real-time PCR and three commonly used algorithmic programs, GeNorm, NormFinder and BestKeeper, were used to evaluate the stability of expression of the candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K , RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) in skeletal muscle-derived satellite cells at 0, 12, 24, 36 and 48 h of growth and after differentiation for 0, 2, 4, 6 and 8 days. The expression of two satellite cell marker genes, CCNA2 and MYF5, was used for validation analysis. The results of the software analyses showed that GAPDH and RPS15A were the most stable reference gene combinations during in vitro proliferation of bovine skeletal muscle-derived satellite cells, RPS15A and RPS9 were the most stable reference gene combinations during in vitro induction of differentiation of the cells, and PPIA was the least stable reference gene during proliferation and differentiation and was not recommended. This study lays the foundation for the selection of reference genes for qRT-PCR during the proliferation and induction of differentiation of bovine skeletal muscle-derived satellite cells.

Analysis of stability of reference genes for qPCR in bovine preadipocytes during proliferation and differentiation in vitro.

The stability of internal reference genes is crucial to the reliability of gene expression results using real-time fluorescence quantitative PCR (qRT-PCR). Inappropriate reference genes may lead to inaccurate results or even wrong conclusions. This study aims to identify stable reference genes for analyzing the expression of proliferation-related and differentiation-inducing genes in bovine primary preadipocytes (BPPs) in vitro. In this study, the stability of 16 candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K, RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) for qRT-PCR at proliferation and differentiation stages of BPPs was investigated by three different algorithms (geNorm, NormFinder and BestKeeper). The expression of two marker genes, PCNA and LPL, was used to determine the validity of the candidate reference genes (RGs) at the proliferation and differentiation stages, respectively. The results showed that GAPDH and RPS15A were the most stable RGs in the proliferation of bovine primary preadipocyte, while PPIA was the least stable internal reference gene. RPLP0 and EIF3K were the most stable RGs in the differentiation induction of bovine primary preadipocyte, while GAPDH was the least stable internal reference gene. This study of RGs laid the foundation for subsequent research into the mechanism of proliferation and differentiation of BPPs in vitro using qRT-PCR.

9-Bromo-2,3-diethylbenzo[de]chromene-7,8-dione (MSN54): A novel non-intercalative topoisomerase II catalytic inhibitor.

Novel mansonone F derivative MSN54 (9-bromo-2,3-diethylbenzo[de]chromene-7,8-dione) exhibited significant cytotoxicity against twelve human tumor cell lines in vitro, with particularly strong potency against HL-60/MX2 cell line resistant to Topo II poisons. MSN54 was found to have IC50 of 0.69 and 1.43 µM against HL-60 and HL-60/MX2 cells, respectively. The resistance index is 10 times lower than that of the positive control VP-16 (etoposide). Various biological assays confirmed that MSN54 acted as a Topo IIα specific non-intercalative catalytic inhibitor. Furthermore, MSN54 exhibited good antitumor efficacy and low toxicity at a dose of 5 mg/kg in A549 tumor xenograft models. Thus, compound MSN54 is a promising candidate for the development of novel antitumor agents.

CNOT1 is involved in TTP­mediated ICAM­1 and IL­8 mRNA decay.

Subunit 1 is the scaffold protein of the carbon catabolite repressor protein 4 (CCR4)­negative on TATA (NOT) complex (CNOT1). In our previous study, it was reported that tristetraprolin (TTP) could recruit subunit 7 of the CCR4­NOT complex (CNOT7) to induce the degradation of intercellular adhesion molecule­1 (ICAM­1) and interleukin­8 (IL­8) mRNA in human pulmonary microvascular endothelial cells (HPMECs). It was additionally demonstrated that TTP, CNOT7 and CNOT1 formed a complex in HPMECs. However, whether CNOT1 is involved in TTP­mediated ICAM­1 and IL­8 mRNA decay remains unclear. The present study demonstrated that CNOT1 knockdown improved ICAM­1 and IL­8 mRNA stabilization and protein expression levels. The immunofluorescence results demonstrated that CNOT1, CNOT7 and TTP are co­localized in the cytoplasm. CNOT1 silencing abolished CNOT7 and TTP coimmunoprecipitation. However, CNOT7 silencing did not influence CNOT1 and TTP coimmunoprecipitation, and TTP silencing additionally did not influence CNOT1 and CNOT7 coimmunoprecipitation. These results together with the authors' previous study, have identified that CNOT1 provides a platform for the recruitment of TTP and CNOT7, and is involved in TTP­mediated ICAM­1 and IL­8 mRNA decay.