Sub Topic | Secondary Topic: Biotherapeutics and Biotechnology - Cell-Based Assay | Functional Response-Pharmacodynamic
Authors: Walid Fayad, National Research Centre, Egypt; Hazem Hassan, University of Maryland (Main Author, Presenting Author)
Presenting Author: Hazem Hassan
Purpose: Despite the enormous development in understanding the molecular basis of malignant tumours, the cure rates of cancers that require systemic treatment are still limited to 4%. Thus, there is a pressing need for the development for more efficient drugs for curing cancer. Most currently used chemotherapeutic agents were identified in cell based cytotoxicity assays where cancer cells are grown as monolayers. It is thought that this type of screen will continue to play an important role in cancer drug discovery. However, it was found that monolayers screening is not necessarily predictive for in vivo activity. Spheroids are superior to monolayers in modeling solid tumors in terms of growth kinetics, gene expression, three dimensional structure, multicellular resistance and similarity of extracellular matrix. Thus, spheroids are considered more efficient in predicting in vivo anticancer activity compared to their monolayers counterpart.
Methods: A library composed of 320 chemically diverse compounds (Bleiswijkseweg 552712 PB Zoetermeer, The Netherlands) was screened for cytotoxicity on MCF7 human breast carcinoma spheroids. Spheroids were treated for 3 hours then media was changed, and were incubated for further 5 days till cytotoxicity assessment. The initial screen of each compound (50 µM) was performed on MCF7 spheroids in triplicates. Compounds that showed cytotoxic activity were identified and then additional dose response studies were performed on both MCF7 spheroids and counter-screened on RPE1 normal cells spheroids at final concentrations: 50, 25, 12.5, 6.25, 3.12 and 1.56 µM in triplicates. The counter-screen aimed to identify compound with selective toxicity towards cancer cells. 2 µM staurosporine was used as positive control, and 0.5% DMSO as negative control. The cytotoxic activity was measured by the acid phosphatase assay. Clonogenic assay was also performed using both MCF7 and RPE1 (normal immortalized human retinal epithelial cell line) spheroids to conduct dose response studies for the identified active compound. After 5 days incubation, the spheroids were washed, trypsinized, gently pipetted and then transferred to 6-well plates. Staurosporine (2 µM) treated spheroids were used as positive control, while 0.5% DMSO treated spheroids were the negative control. All treatments were performed in triplicates. After 13 days incubation, clonogenicity percent reduction was calculated compared to the negative control.
Results: One compound, (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate, showed ≥80% cytotoxic activity at 50 µM. On performing a dose response study on MCF7 and RPE1 spheroids, the IC50 values were 30.9 and 30.3 µM, respectively, indicating non-selectivity towards cancer spheroids especially at relatively higher concentrations. However, the dose response study and colonogenic assay both performed on the two cell lines spheroids indicated that at relatively lower concentration of 6.25 µM, the identified compound possessed preferential cytotoxicity towards the cancer cell line and negligible activity towards the normal cells.
Conclusion: The cytotoxicity profile of (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate on both cancer and normal cells spheroids demonstrates it as a promising anticancer agent with greater selective activity towards cancer cells at lower concentrations. Further mechanistic, metabolism, transport and in-vivo PK/PD studies are warranted to fully characterize this compound.
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