Recombinant Spinach Ribosome-Inactivating Protein Induces Cytotoxicity and Alters Gene Expression Associated with PTEN/AKT/mTOR Signaling, Apoptosis, and Stemness in HT29 cells.

Abstract

Colorectal cancer (CRC) remains a major therapeutic challenge, highlighting the need for novel anticancer strategies. These challenges have increased interest in bioactive natural molecules with anticancer potential. In this study, we investigated the antitumor activity of a recombinant spinach (Spinacia oleracea) type I ribosome-inactivating protein (SoRIP) in HT29 colorectal cancer cells and evaluated its effects on cellular survival, apoptosis, and stemness-associated pathways. The SoRIP coding sequence was cloned, heterologously expressed in Escherichia coli BL21 (DE3), and purified by Ni-affinity chromatography, yielding a recombinant protein of approximately 22 kDa. Recombinant SoRIP reduced HT29 cell viability in a concentration-dependent manner after 48 h of treatment, with an IC50 of 48 μg/mL and cell viability of 51.3 ± 2.5% at this concentration. Annexin V-FITC/PI flow cytometry demonstrated a concentration-dependent increase in total apoptotic cells, reaching approximately 15%, 58%, and 79% following exposure to 12, 48, and 180 μg/mL SoRIP, respectively. At the transcriptional level, SoRIP increased CASP3 expression by up to 3.8-fold and PTEN expression by up to 4.3-fold, while reducing AKT and mTOR expression to 0.60- and 0.70-fold, respectively, at 48 μg/mL. BCL2 and CD44 expression were also reduced to approximately 0.65- and 0.70-fold, respectively. These findings indicate that recombinant SoRIP is associated with reduced cell viability, enhanced apoptosis, and coordinated transcriptional modulation of PTEN/AKT/mTOR-related, apoptosis-associated, and stemness-associated genes in HT29 cells. Collectively, these results support SoRIP as a candidate for further investigation in the HT29 colorectal cancer model.