Evaluating the impact of Apoptosis and ferroptosis induction, using supression of AKT1 and GPX4 genes expression, in Oxaliplatin resistancy in Colorectal cancer cell line

Abstract

AKT1 and GPX4 are key regulators of Apoptosis and Ferroptosis pathways. Inhibition of these genes might induce the eradication of Oxaliplatin resistance in colorectal cancer cell lines. Purpose: Induction of different kind of cell death to increasing sensitivity or overcoming resistance to Oxaliplatin in colon cancer cells; an in-vitro study Procedure: Resistance HT-29 cell lines, was cultured on RPMI media containing 10% FBS, at 37oC, in humidified incubator, containing 5% CO2. Both cell lines treated with Oxaliplatin with/without gene silencing procedures as follow: For gene silencing experiments, designed GPX4 and AKT1 SiRNAs transformed using Hyperfect kit. Afterward, silencing procedure confirmed with gene expression analysis using Real-time PCR and western bloting methods. Subsequently, Apoptosis induction evaluated using Annexin-PI Flowcytometry. Afterward, for evaluation of Ferroptosis induction, total cellular Lipidic ROS level analyzed using Flowcytometry technique. Finally, the cellular viability in coadministration of oxaliplatin and siRNAs evaluated using Flowcytometry by trypan blue exclusion assay. Findings: Real-time PCR and western bloting techniques was used for confirmation of gene silencing in HT-29 resistance cells. Flowcytometry analysis reveal that more than 70% of AKT1 siRNA treated cells enter into the apoptotic cell death. Similarly, more than 80% of GPX4 siRNA trated cells stained positive for total cellular Lipidic ROS using Ferroptosis technique. Finally, the combined administration of Oxaliplatin and either Apoptosis or Ferroptosis inducer siRNAs shown that either AKT1 or Gpx4 gene silencing have high potential in eradicating the resistance cell line. Conclusion: Ferroptosis can be substitute strategy instead of Apoptosis to eradicate chemotherapy resistance cancer cells.