Background: Piperine, an alkaloid from Piper nigrum, modulates oxidative stress, proliferation, and survival pathways in several cancer models; however, its mechanistic effects in colorectal epithelial Caco-2 cells remain insufficiently defined. Objective: This study aimed to investigate the cytotoxic, antiproliferative, oxidative, autophagic, and anti-migratory effects of piperine in Caco-2 cells. Methods: Caco-2 cells were treated with piperine (0.001–0.1 mg/mL) for up to 72 h. Cell viability, proliferation, and migration were assessed using SRB and scratch assays. Oxidative stress, apoptosis, autophagy, and tight junction integrity were evaluated through ROS quantification, Western blotting, gene expression analysis, confocal microscopy, and transmission electron microscopy (TEM). NACET was used to determine the contribution of oxidative stress to piperine-induced cytotoxicity and autophagy. Results: Piperine induced a time- and dose-dependent reduction in viability, with viability decreasing to 53.0 ± 2.88% at 0.1 mg/mL after 72 h. Proliferation decreased to 51% of control levels (p < 0.001), accompanied by p21 upregulation (p < 0.05), indicating G2/M cell cycle arrest. Piperine markedly increased intracellular ROS (p < 0.001), downregulated NRF2 (p < 0.05), and suppressed GSTA1 expression (p < 0.001), while NACET co-treatment restored viability (p < 0.001). No activation of caspase-dependent apoptosis was observed. Piperine significantly enhanced autophagic flux, as shown by the increased LC3B-II/LC3B-I ratio (p < 0.01), elevated LC3B-II/LAMP-1 co-localization (p < 0.01), and chloroquine-induced accumulation of LC3B-II and p62 (p < 0.01), with preserved lysosomal function. TEM analysis confirmed a marked increase in double-membrane autophagosomes in piperine-treated cells compared with controls. NACET reduced LC3B-II/LC3B-I levels, increased p21 expression, and significantly improved cell viability, indicating that piperine-induced autophagy is cytotoxic and driven by oxidative stress. Additionally, piperine upregulated occludin (p < 0.01) and reduced cell migration independently of proliferation (p < 0.01). Conclusions: Piperine exerts antiproliferative effects in Caco-2 cells through ROS-mediated stress, p21-dependent G2/M arrest, and activation of cytotoxic autophagy. Its ability to impair migration and enhance tight junction integrity further supports its potential as a complementary therapeutic agent in colon cancer.

Capsaicin, the principal bioactive compound of chilli peppers, has emerged as a promising natural molecule with anticancer potential in several malignancies, including colorectal cancer. However, the molecular mechanisms underlying its effects in colorectal cancer cells remain incompletely understood. In the present study, we investigated the effects of capsaicin on Caco-2 human colorectal adenocarcinoma cells, focusing on cell viability, proliferation, migration, cell-cycle regulation, and autophagy. Capsaicin (50–200 µM) reduced cell viability in a dose- and time-dependent manner and significantly inhibited proliferation and migratory capacity. Capsaicin induced G2/M cell-cycle arrest, accompanied by increased p21 expression and reduced p53 phosphorylation. Notably, capsaicin-induced cytotoxicity was not associated with activation of caspase-dependent apoptosis and was instead associated with marked accumulation of autophagic structures and increased LC3B-II/LAMP-1 co-localization. Chloroquine co-treatment failed to further increase LC3B-II or p62 accumulation, indicating that autophagic degradation was already impaired. Although lysosomal abundance remained unchanged, capsaicin significantly reduced mature cathepsin-D protein, demonstrating compromised lysosomal degradative capacity and defective autophagic flux. In addition, capsaicin upregulated occludin expression, suggesting enhanced tight-junction integrity and reduced migratory potential. Collectively, these findings, at least explorative, demonstrate that capsaicin reduces Caco-2 cell viability and induces G2/M cell-cycle arrest, accompanied by p21 upregulation and impaired lysosomal degradative capacity, highlighting disruption of the autophagy–lysosomal pathway as a potential mechanism underlying its anticancer activity in Caco-2 cells.