In vivo, In vitro, and In silico Studies of Umbelliferone and Irinotecan on MDA-MB-231 Breast Cancer Cell Line and Drosophila melanogaster Larvae.

Aims: Deaths from cancer are still very common all over the world and continue to be the focus of scientific research. Chemotherapy is one of the primary treatments used to prevent deaths from cancer. Side effects of chemotherapeutic drugs and resistance of cells to drugs are essential problems that limit the treatment process. Drug combination therapy is regarded as a significant application that inhibits the growth of tumors and is anticipated to provide a solution for the issues encountered. The combination therapy aims at a synergistic effect that will limit drug resistance and cytotoxic effects with appropriate drug combinations. In this context, we aim to investigate the in vitro, in vivo, and in silico effects of single and combined doses of umbelliferone and irinotecan, known for their anticarcinogenic and curative effects, on MDA-MB-231 breast cancer cell lines and the model organism Drosophila melanogaster.

Background: Irinotecan is currently used as an anticarcinogenic drug. Anticarcinogenic effects of umbelliferone have also been detected. The in vivo, in vitro, and in silico impacts of single and combined doses use of these two agents are not yet available in the literature.

Objective: This study aims to determine the anticarcinogenic effects of single and combined use of umbelliferone and irinotecan at the molecular level. It also attempts to determine the binding energies of chemicals to cancerrelated proteins through docking and molecular dynamic studies.

Method: The cytotoxic effects of individual and combinational doses of umbelliferone and irinotecan on the MDAMB- 231 cell line and D. melanogaster were calculated by XTT and probit analyses. IC50 values for the cancer cells, LC50, and LC99 values for D. melanogaster were found. Gene expression analysis was performed to determine the effects of chemical agents on miR-7, miR-11, and miR-14, and their expression levels were found. The sequences of miRNAs not found in the literature were determined, and their molecular imaging was performed. In addition, the binding energies of irinotecan and umbelliferone to Bcl-2, Bad, and Akt1 proteins, which are known to have apoptotic effects, were found by the molecular docking method. Molecular dynamics studies of Bad proteins and chemicals were also performed. The drug potential of chemicals was determined by ADME/T analysis.

Result: The cytotoxic effect on cells was calculated, and the IC50 value of umbelliferone was calculated as 158 μM, the IC50 value of irinotecan was calculated as 48,3 μM and the IC50 value was calculated as 20 μM. In the probit analysis performed to calculate the cytotoxic effects of drugs on D. melanogaster, the LC50 value of umbelliferone was 2,5 μM, and the LC99 value was 13,4 μM. The LC50 value of irinotecan was found to be 0,1 μM, and the LC99 value was 0,28 μM. It was concluded that single and combined doses of chemicals in the invasion experiment significantly affected the spread of cells. As a result of expression analysis, a significant increase in HsamiR- 7 (Homo sapiens miRNA-7), Hsa-miR-14 (Homo sapiens miRNA-14), and Hsa-miR-11(Homo sapiens miRNA-11) expression was observed in cells treated with umbelliferone irinotecan compared to the control groups.

Conclusion: In our study, it can be concluded that the cytotoxic effects of individual and combination doses of umbelliferone and irinotecan on MDA-MB-231 cells and D. melanogaster larvae are significant. In addition, the effects of umbelliferone and irinotecan on the expression level of miR-7, which is a common D. melanogaster and human miRNA, should be widely investigated. Expression analyses and docking studies of Hsa-miR-11 and Hsa-miR-14, which have been newly studied and are not in data repositories, are important for cancer research. In particular, the expression and binding energy of these miRNAs in new drug combinations and the expression level in different cancer cell lines are important for future studies. Another crucial point is that in vivo tests using different model species validate the usage of drugs at both single and mixed dosages. Other: As a result of this study, the in vivo, in vitro, and in silico effects of single and combined doses of umbelliferone and irinotecan were determined. In future studies, it would be useful to determine the binding energies of umbelliferone and irinotecan to other cancer-related proteins and to find their interactions with different miRNAs. Additionally, studies on different model organisms are also important.