Quercetin and a plant substance, identified using a multi-copy number of DNA topoisomerase I in recombinant Pichia pastoris, inhibited MDA-MB-231 proliferation via different manners.

The study employed an in vivo strategy to construct a multi-copy number of human DNA topoisomerase I (hTopI) gene using pPIC3.5K vector in GS115 strain of Pichia. The yeast transformant (GS115-pPIC3.5K-hTopI; clone) was then used to investigate the preliminary growth effect of a pure compound (quercetin) and a standardised subfraction of ethanolic red onion peel extract (F1). The clones’ cell density was likely to be unaffected; only the total protein expression and enzyme activity were increased following the increased copy number of hTop1 in the host. The clone that showed the target enzyme's highest activity is said to respond specifically to growth inhibitors, whereby both quercetin and F1 were proven to be potential growth inhibitors as assessed by the MTT assay. In the process, quercetin reduced cell proliferation by inducing apoptosis and cell cycle arrest (S phase only), whereas F1 reduced cell proliferation by inducing cell cycle arrest only (S and G2 phases). Quercetin and F1 induced CYP1A1 and CYP1B1 (carcinogenicity) gene mRNA expression, but only F1 induced CYP2S1 (cytotoxicity) gene mRNA expression in the treated cells, suggesting that both quercetin and F1 inhibited the cell proliferation of MDA-MB-231 via different manners. The newly developed GS115-pPIC3.5K-hTopI can be used to select various potential substances for breast cancer treatment in the future.

Genome-wide proximity between RNA polymerase and DNA topoisomerase I supports transcription in Streptococcus pneumoniae

Streptococcus pneumoniae is a major cause of disease and death that develops resistance to multiple antibiotics. DNA topoisomerase I (TopoI) is a novel pneumococcal drug target. TopoI is the sole type-I pneumococcal topoisomerase that regulates supercoiling homeostasis in this bacterium. In this study, a direct in vitro interaction between TopoI and RNA polymerase (RNAP) was detected by surface plasmon resonance. To understand the interplay between transcription and supercoiling regulation in vivo, genome-wide association of RNAP and TopoI was studied by ChIP-Seq. RNAP and TopoI were enriched at the promoters of 435 and 356 genes, respectively. Higher levels of expression were consistently measured in those genes whose promoters recruit both RNAP and TopoI, in contrast with those enriched in only one of them. Both enzymes occupied a narrow region close to the ATG codon. In addition, RNAP displayed a regular distribution throughout the coding regions. Likewise, the summits of peaks called with MACS tool, mapped around the ATG codon in both cases. However, RNAP showed a broader distribution towards ATG-downstream positions. Remarkably, inhibition of RNAP with rifampicin prevented the localization of TopoI at promoters and, vice versa, inhibition of TopoI with seconeolitsine prevented the binding of RNAP to promoters. This indicates a functional interplay between RNAP and TopoI. To determine the molecular factors responsible for RNAP and TopoI co-recruitment, we looked for DNA sequence motifs. We identified a motif corresponding to a -10-extended promoter for TopoI and for RNAP. Furthermore, RNAP was preferentially recruited to genes co-directionally oriented with replication, while TopoI was more abundant in head-on genes. TopoI was located in the intergenic regions of divergent genes pairs, near the promoter of the head-on gene of the pair. These results suggest a role for TopoI in the formation/stability of the RNAP-DNA complex at the promoter and during transcript elongation.

Case Report: 7-Ethyl-10-Hydroxycamptothecin, a DNA Topoisomerase I Inhibitor, Performs BRD4 Inhibitory Activity and Inhibits Human Leukemic Cell Growth

7-Ethyl-10-hydroxycamptothecin (SN-38) is an active metabolite of CPT-11, which can inhibit DNA topoisomerase I, DNA synthesis and cause frequent DNA single-strand breaks. In our study, SN-38 was characterized as a potent and reversible BRD4 inhibitor [IC50 = 660.2 nM against BRD4 (BD1) and IC50 = 547.7 nM against BRD4 (BD2)] in biochemical assay using drug repurposing strategy. Additional cellular assay suggested that SN-38 can bind BRD4 in human leukemic cell K562 and inhibit cell growth with IC50 = 0.2798 μM in a BRD4 dependent manner partially. Additionally, mechanism study indicated that SN-38 can induce the accumulation of BRD4 substrate c-Myc and cleavage of caspase 3. In sum, our findings identified BRD4 as a new target of SN-38 and reveals SN-38 as a modifier of histone acetylation reader for the first time, which may provide a new insight for further optimization of dual target inhibitor.

Quercetin and a plant substance, identified using a multi-copy number of DNA topoisomerase I in recombinant Pichia pastoris, inhibited MDA-MB-231 proliferation via different manners.

The study aimed to employ an in vivo strategy to construct a multi-copy number of human DNA topoisomerase I (hTopI) gene using pPIC3.5K vector in GS115 strain of Pichia. The yeast transformant (GS115-pPIC3.5K-hTopI) was then used to investigate the preliminary growth effect of a pure compound (quercetin) and a standardised subfraction of ethanolic red onion peel extract (F1). The underlying mechanisms of quercetin and F1 were then tested on MDA-MB-231 for the cell cycle profile and apoptosis by flow cytometry, and the mRNA expression of CYP genes by real-time PCR. His + yeast transformants (clones) with multi-copy inserts resistant to various concentrations of Geneticin were successfully selected in the study. The clones’ cell density was likely to be unaffected; only the total protein expression and enzyme activity were increased following the increased copy number of hTop1 in the host. The clone that showed the target enzyme's highest activity is said to respond specifically to growth inhibitors, whereby both quercetin and F1 were proven to be potential growth inhibitors as assessed by the MTT assay. In the process, quercetin reduced cell proliferation by inducing apoptosis and cell cycle arrest (S phase only), whereas F1 reduced cell proliferation by inducing cell cycle arrest only (S and G2 phases). Quercetin and F1 induced CYP1A1 and CYP1B1 (carcinogenicity) gene mRNA expression, but only F1 induced CYP2S1 (cytotoxicity) gene mRNA expression in the treated cells, suggesting that both quercetin and F1 inhibited the cell proliferation of MDA-MB-231 via different manners. The newly developed GS115-pPIC3.5K-hTopI can be used to select various potential substances for breast cancer treatment in the future.

Why Should DNA Topoisomerase I Have a Scaffold Activity?

Since the early 1990s, in vitro studies have demonstrated that DNA topoisomerase I promotes RNA polymerase II transcription, acting as a cofactor, regardless of its catalytic activity. Recent studies, carried in vivo, using yeast as a model system, also demonstrate that DNA topoisomerase I is able to recruit, without the involvement of its catalytic activity, the Sir2p deacetylase on ribosomal genes thus contributes to achieve their silencing. In this review, the DNA topoisomerase I capability, acting as a scaffold protein, as well as its involvement and role in several macromolecular complexes, will be discussed, in light of several observations reported in the literature, pointing out how its role goes far beyond its well-known ability to relax DNA.