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.

DNA topoisomerases are essential enzymes that control the topological state of DNA replication during mitosis. These enzymes are classified based on their mechanisms and physical properties. During mitosis, superhelical DNA must be unwound or relaxed by DNA topoisomerases prior to a decoding step by DNA processing enzymes, such as DNA polymerase and RNA polymerase. By blocking the reaction of resealing the breaks in the DNA ultimately can result in cellular death. Compounds that inhibit the catalytic function of these enzymes can serve as potential anticancer agents. DNA topoisomerases are found in nature and used as high quality and well-validated targets for the screening of potential anticancer agents. Our current work focuses on determining potential anticancer agents from natural resources using DNA topoisomerases as the screening targets. Large scale production of these enzymes using recombinant DNA technology in our academic laboratory is utilised to avoid dependence on expensive commercially available enzymes. The in-house produced enzymes can also be used to enhance our research in the field of molecular medicine by providing an enzyme source that can be used to screen potential anticancer agents, and for other newly developed diagnostic and medical research projects in the near future as well as a step in moving our efforts into the industrial sector.

The present study aimed to employ an in vivo strategy for the establishment of multi-copy gene constructs of human DNA topoisomerase I (hTopI) using the pPIC3.5K vector in Pichia. The clones with multi-copy inserts (His + transformants) that were able to survive in the highest concentration of Geneticin ® were found to express the highest expression level of total protein and exhibited the highest target enzyme activity. The highest level of total protein found was 1.76 mg/ml in GS115-pPIC3.5K-hTop1, which was resistant to 1.00 mg/ml Geneticin at 48 h of incubation. The highest enzyme activity of hTopI was also observed in the culture expressed by GS115-pPIC3.5K-hTopI, which was resistant to 1.00 mg/ml Geneticin ® (19.7x10 4 Ul/OD 600 ). On the whole, the present study provides information regarding the production of target protein from recombinant Pichia using only a shaker flask system, which can be further developed as an in-house resource for screening potential anticancer agents.

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.