Dense granule protein 16 (GRA16), an effector protein from Toxoplasma gondii, has been reported to enhance antitumor responses in several malignancies. However, whether GRA16 can enhance the inhibitory effects of standard anticancer drugs across diverse cancer types remains unclear. Gemcitabine, oxaliplatin, and irinotecan continue to play a key role in clinical oncology; however, their efficacy is limited by persistent tumor survival-promoting pathways. In this study, we stably expressed GRA16 in HepG2, PANC-1, and HCT116 cells and evaluated their responses to gemcitabine, oxaliplatin, and irinotecan under 50% inhibitory concentration-normalized dosing conditions. In vector control cells, baseline drug sensitivity was quantified using the CCK-8 assay across 0–64 μM after 48 h of exposure, and 50% inhibitory concentration was determined for each drug and cell line. Time-dependent proliferation assays (0–72 h) demonstrated that GRA16 reduced the basal growth rate and enhanced the growth inhibitory effects of all 3 drugs in all 3 cell models. Representative microscopy images at 48 h were consistent with reduced cell density in GRA16-expressing cultures under each treatment condition. These in vitro data indicate that GRA16 enhances the inhibitory effects of standard chemotherapeutic agents in hepatic, pancreatic, and colorectal cancer cell lines. This study provides a cross-cancer, multi-drug evaluation of GRA16-associated enhancement of drug response and includes, to our knowledge, the first assessment in a pancreatic cancer model. The underlying mechanism may involve pathway modulation, as suggested by previous studies and supported by the pancreatic cancer model although it was not comprehensively examined across all cell lines.

Colorectal cancer (CRC) with KRAS mutations represents a clinically challenging subgroup with variable treatment responses. Among these tumors, codon 12 mutations are associated with poor prognosis and treatment resistance, while KRASG12D-mutant CRCs lack effective mutation-specific therapeutic options. Toxoplasma gondii dense granule protein 16 (GRA16) is a parasite-derived host-modulating effector that modulates phosphatase and tensin homolog (PTEN)/p53, protein phosphatase 2A-B55 (PP2A-B55)-associated signaling, and cancer-associated metabolic pathways. We evaluated the anticancer effects of GRA16 in KRASG12D-mutant LS174T cells using KRASG13D-mutant HCT116 cells as a comparative TP53-wild-type CRC model. Stable GRA16-expressing cells were generated by lentiviral transduction and assessed for proliferation, cell death-associated responses, DNA damage/apoptosis-related protein markers, key signaling proteins, and targeted transcriptional changes. GRA16 inhibited proliferation and increased cell death-associated populations in both cell lines, accompanied by elevated γ-H2A.X, cleaved PARP, and cleaved caspase-3 levels. In HCT116 cells, GRA16 increased PTEN, p53, and PP2A-B55 protein levels and reduced total AKT and Survivin proteins, together with BAX induction and suppression of cell-cycle/checkpoint-, epithelial mesenchymal transition/motility-, and glycolysis-associated transcripts. In LS174T cells, GRA16 increased PP2A-B55 and reduced Survivin protein levels, whereas PTEN protein was unchanged, p53 was reduced, and total AKT was increased. These changes were accompanied by limited p53/BAX-associated transcriptional engagement, glycolytic attenuation, shared cell-cycle/checkpoint-associated gene suppression, and stress-adaptive remodeling. These findings suggest that GRA16 induces cell line-dependent anticancer-associated responses in KRAS-mutant CRC cells, with a prominent PTEN/p53-associated response in HCT116 cells and preserved PP2A-B55 induction and Survivin suppression in LS174T cells.