Theaflavins Inhibit Proliferation and Glycolysis of Colorectal Cancer Cells by Downregulating DDIT4

Abstract

Colorectal cancer (CRC) is a common malignancy often characterized by metastasis and poor prognosis. This study attempts to ascertain the anticancer impacts of theaflavin (TF) on CRC cells and examine the fundamental molecular mechanisms, focusing on the function of DDIT4 in CRC progression. This study utilized RNA sequencing for gene expression profiling, differential expression analysis, and Venn diagram analysis for overlapping genes. Protein interactions were explored, while cell viability was evaluated using colony formation assays and Cell Counting Kit-8 (CCK-8). Flow cytometry was employed for apoptosis analysis, and Transwell assays measured cell migration and invasion. ATP synthesis, lactate production, and glucose uptake were analyzed to evaluate metabolic changes, with protein and RNA expression identified by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). This study reveals that TF effectively inhibits CRC cell invasion, migration, and proliferation in a dose- and time-dependent manner. TF induces apoptosis by suppressing the antiapoptotic protein Bcl-2 and enhancing proapoptotic proteins (Cleaved Caspase-3, Bax, and Caspase-9). Through bioinformatics analysis, DDIT4 was identified as a key target gene. Additionally, correlation analysis highlighted a positive relationship between DDIT4 and the glycolysis/gluconeogenesis pathway. TF downregulates DDIT4 expression, which suppresses CRC cell proliferation and glycolysis. Moreover, DDIT4 overexpression partially reverses the suppressive impacts of TF on glycolysis and cell viability. These observations imply that TF suppresses CRC progression by targeting DDIT4 and regulating glycolytic activity, highlighting its promise as a medicinal substance for the treatment of CRC.