Potential use of Pichia pastoris strain SMD1168H expressing DNA topoisomerase I in the screening of potential anti‑breast cancer agents

Abstract

Cancer chemotherapy possesses high toxicity, particularly when a higher concentration of drugs is administered to patients. Therefore, searching for more effective compounds to reduce the toxicity of treatments, while still producing similar effects as current chemotherapy regimens, is required. Currently, the search for potential anticancer agents involves a random, inaccurate process with strategic deficits and a lack of specific targets. For this reason, the initial in vitro high-throughput steps in the screening process should be reviewed for rapid identification of the compounds that may serve as anticancer agents. The present study aimed to investigate the potential use of the Pichia pastoris strain SMD1168H expressing DNA topoisomerase I (SMD1168H-TOPOI) in a yeast-based assay for screening potential anticancer agents. The cell density that indicated the growth of the recombinant yeast without treatment was first measured by spectrophotometry. Subsequently, the effects of glutamate (agonist) and camptothecin (antagonist) on the recombinant yeast cell density were investigated using the same approach, and finally, the effect of camptothecin on various cell lines was determined and compared with its effect on recombinant yeast. The current study demonstrated that growth was enhanced in SMD1168H-TOPOI as compared with that in SMD1168H. Glutamate also enhanced the growth of the SMD1168H; however, the growth effect was not enhanced in SMD1168H-TOPOI treated with glutamate. By contrast, camptothecin caused only lower cell density and growth throughout the treatment of SMD1168H-TOPOI. The findings of the current study indicated that SMD1168H-TOPOI has similar characteristics to MDA-MB-231 cells; therefore, it can be used in a yeast-based assay to screen for more effective compounds that may inhibit the growth of highly metastatic breast cancer cells.

Yeast-Based Screening of Anti-Viral Molecules

“…It is important to mention that certain yeasts, especially S. cerevisiae followed by S. pombe , remain the most used species in viral screenings. Surprisingly, other yeasts such as Komagataella phaffii (formerly Pichia pastoris ) are rarely used for screening purposes [ 132 ], even though this yeast species has already gained significant importance as a biological model [ 133 , 134 , 135 , 136 ]. Several molecules selected through yeast-based screening are currently in use (e.g., statin), which increases the confidence in and reliability of using this approach.…”

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 preliminary screening of anticancer agents using cell-based assays is not encouraging because the assays are costly and require speci c equipment, expertise, and specialised facilities. Therefore, it is better to have other alternatives to the screening process rather than relying on expensive and commercially available cell-based assays alone [16]; only candidate compounds that exhibit a positive effect at the early discovery stages will then proceed to the next drug discovery steps, to faster the preliminary screening process. In this study, a multicopy number of the hTopI gene was produced using the pPIC3.5K vector in the GS115 strain of Pichia to enhance the protein yield of interest in the host.…”