Investigation of the Anticancer Mechanism of Isoorientin Isolated from Eremurus Spectabilis Leaves via Cell Cycle Pathways in HT-29 Human Colorectal Adenocarcinoma Cells.

Objective: Isoorientin (ISO) is a flavonoid compound extracted from plant species. The goal of this study was to determine the potential antiproliferative effects of ISO in HT-29 human colorectal adenocarcinoma cell line in vitro , specifically on cell viability, apoptosis, and cell cycle pathways.

Materials and Methods: The cytotoxic effect of ISO isolated from E. spectabilis was measured using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in HT-29 cell lines. Total RNA was isolated using Tri-Reagent protocol. The effects of ISO on apoptosis-related gene were detected using real-time polymerase chain reaction (RT-PCR). The findings were analyzed using "Delta-Delta CT" ΔΔCT method and evaluated using a computer program. Volcano plot analysis was used for comparing groups and the data obtained were statistically analyzed using Student t test.

Results: According to XTT result analysis, the 50% inhibitory concentration (IC50) value of ISO was 125 μM at the 48th h in HT-29 cells. The RT-PCR analysis in HT-29 cells showed that Cyclin D1 ( CCND1 ), Cyclin-dependent kinase 6 ( CDK6 ), BAX, BCL-2 , Checkpoint kinase 1-2 (CHEK1, CHEK2) and Excision repair cross-complementing 1 ( ERCC1 ) expressions were reduced in ISO-treated cells compared with those in the control group of cells. P53, P21, Caspase-3 (CASP-3) , Caspase-8 (CASP-8) , and Caspase-9 ( CASP-9 ) gene expressions were increased Ataxia Telengiectasia and Rad-3 related ( ATR ) was activated in the ISO-treated group of cells compared with those in the control group of cells (p<0.05).

Conclusion: ISO affected the proliferation of colorectal cancer (CRC) cells via cell cycle pathways. It also altered apoptosis gene expression. These results demonstrated that ISO can be a therapeutic agent for CRC treatment; however, more studies are needed to investigate its mechanism of actions.