TY - JOUR

T1 - Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo

AU - Mohamed, Islam Saber Ead

AU - Sen’kova, Aleksandra V.

AU - Markov, Oleg V.

AU - Markov, Andrey V.

AU - Savin, Innokenty A.

AU - Zenkova, Marina A.

AU - Mironova, Nadezhda L.

N1 - Funding Information: Author Contributions: Conceptualization, N.L.M., A.V.S. and M.A.Z.; methodology, A.V.S., A.V.M., A.V.M., O.V.M. and N.L.M.; software, O.V.M., A.V.S. and A.V.M.; validation, I.S.E.M., A.V.M. and A.V.S.; formal analysis, N.L.M., A.V.S., I.A.S. and A.V.M.; investigation, I.S.E.M., A.V.S., O.V.M., formal analysis, N.L.M., A.V.S., I.A.S. and A.V.M.; investigation, I.S.E.M., A.V.S., O.V.M., A.V.M. and A.V.M. and I.A.S.; resources, M.A.Z.; data curation, N.L.M. and M.A.Z.; writing—original draft I.A.S.; resources, M.A.Z.; data curation, N.L.M. and M.A.Z.; writing—original draft preparation, preparation, I.S.E.M., A.V.S., A.V.M. and N.L.M.; writing—review and editing, M.A.Z.; visualiza-I.S.E.M., A.V.S., A.V.M. and N.L.M.; writing—review and editing, M.A.Z.; visualization, O.V.M. and tion, O.V.M. and A.V.S.; supervision, N.L.M. and M.A.Z.; project administration, N.L.M.; funding A.V.S.; supervision, N.L.M. and M.A.Z.; project administration, N.L.M.; funding acquisition, M.A.Z. acquisition, M.A.Z. All authors have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Russian Science Foundation (grant no. 19-74-30011) and Funding: This research was funded by the Russian Science Foundation (grant no. 19-74-30011) and the Russian Government funded budget project of ICBFM SB RAS no. 121031300044-5. I.S.E.M. was the Russian Government funded budget project of ICBFM SB RAS no. 121031300044-5. I.S.E.M. was funded by the joint executive program between Egypt and Russia (EGY-6507/17). funded by the joint executive program between Egypt and Russia (EGY-6507/17). Institutional Review Board Statement: All animal procedures were carried out in strict accordance Institutional Review Board Statement: All animal procedures were carried out in strict accordance with the approved protocol and recommendations for proper use and care of laboratory animals with the approved protocol and recommendations for proper use and care of laboratory animals (ECC Directive 2010/63/EU). The experimental protocols were approved by the Committee on the (ECC Directive 2010/63/EU). The experimental protocols were approved by the Committee on the Ethics of Animal Experiments with the Institute of Cytology and Genetics SB RAS (ethical approval Ethics of Animal Experiments with the Institute of Cytology and Genetics SB RAS (ethical approval no. 50 dated 23 May 2019). no. 50 dated 23 May 2019). Data Availability Statement: Data are contained within the article or supplementary materials. Data Availability Statement: Data are contained within the article or supplementary materials. Acknowledgments: The authors thank A. Vladimirova for cell maintenance. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/6

Y1 - 2022/6

N2 - In this investigation, we extensively studied the mechanism of antitumor activity of bovine pancreatic RNase A. Using confocal microscopy, we show that after RNase A penetration into HeLa and B16 cells, a part of the enzyme remains unbound with the ribonuclease inhibitor (RI), resulting in the decrease in cytosolic RNAs in both types of cells and rRNAs in the nucleoli of HeLa cells. Molecular docking indicates the ability of RNase A to form a complex with Ku70/Ku80 heterodimer, and microscopy data confirm its localization mostly inside the nucleus, which may underlie the mechanism of RNase A penetration into cells and its intracellular traffic. RNase A reduced migration and invasion of tumor cells in vitro. In vivo, in the metastatic model of melanoma, RNase A sup-pressed metastases in the lungs and changed the expression of EMT markers in the tissue adjacent to metastatic foci; this increased Cdh1 and decreased Tjp1, Fn and Vim, disrupting the favorable tumor microenvironment. A similar pattern was observed for all genes except for Fn in metastatic foci, indicating a decrease in the invasive potential of tumor cells. Bioinformatic analysis of RNase-A-susceptible miRNAs and their regulatory networks showed that the main processes modulated by RNase A in the tumor microenvironment are the regulation of cell adhesion and junction, cell cycle regulation and pathways associated with EMT and tumor progression.

AB - In this investigation, we extensively studied the mechanism of antitumor activity of bovine pancreatic RNase A. Using confocal microscopy, we show that after RNase A penetration into HeLa and B16 cells, a part of the enzyme remains unbound with the ribonuclease inhibitor (RI), resulting in the decrease in cytosolic RNAs in both types of cells and rRNAs in the nucleoli of HeLa cells. Molecular docking indicates the ability of RNase A to form a complex with Ku70/Ku80 heterodimer, and microscopy data confirm its localization mostly inside the nucleus, which may underlie the mechanism of RNase A penetration into cells and its intracellular traffic. RNase A reduced migration and invasion of tumor cells in vitro. In vivo, in the metastatic model of melanoma, RNase A sup-pressed metastases in the lungs and changed the expression of EMT markers in the tissue adjacent to metastatic foci; this increased Cdh1 and decreased Tjp1, Fn and Vim, disrupting the favorable tumor microenvironment. A similar pattern was observed for all genes except for Fn in metastatic foci, indicating a decrease in the invasive potential of tumor cells. Bioinformatic analysis of RNase-A-susceptible miRNAs and their regulatory networks showed that the main processes modulated by RNase A in the tumor microenvironment are the regulation of cell adhesion and junction, cell cycle regulation and pathways associated with EMT and tumor progression.

KW - antitumor activity

KW - EMT markers

KW - intracellular localization

KW - Ku70/Ku80

KW - miRNA

KW - ribonuclease inhibitor

KW - RNase A

UR - http://www.scopus.com/inward/record.url?scp=85132302956&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/5181e40d-71df-311b-b1a6-e9419c2ca639/

U2 - 10.3390/pharmaceutics14061173

DO - 10.3390/pharmaceutics14061173

M3 - Article

C2 - 35745743

AN - SCOPUS:85132302956

VL - 14

JO - Pharmaceutics

JF - Pharmaceutics

SN - 1999-4923

IS - 6

M1 - 1173

ER -