Thymoquinone and pentoxifylline modulate 5-fluorouracil-induced senescence and apoptosis in colorectal cancer spheroids

Background and purpose: Targeting senescent tumor cells with senotherapeutic agents represents a novel therapeutic strategy in overcoming chemotherapy resistance during 5-fluorouracil (5-FU) treatment of colorectal cancer (CRC). This study aimed to evaluate the senotherapeutic potential of thymoquinone (TQ) and pentoxifylline (PTX) in a 3D spheroid model of CRC subjected to 5-FU-induced senescence. Experimental approach: Caco-2 spheroids were generated via the hanging drop method on Poly-HEMA-coated plates with conditioned medium derived from HFF cells. Spheroids were treated for 24 h with 5-FU (60 μΜ; 50% of IC 50 ), TQ (30 μΜ; 50% of IC 50 ), and PTX (3.5 mM; 50% of IC 50 ). Cell viability was measured using the MTT assay. ROS and IL-8 levels were measured. Cell death and apoptosis were evaluated using Calcein-AM and Annexin V/PI assays, and western blotting was used to assess the expression of SASP and apoptosis-associated proteins. β-galactosidase activity was measured as a marker of senescence. Findings/Results: 5-FU effectively induced senescence and apoptosis in Caco-2 spheroids, as evidenced by an increased expression of p53, p16, p21, and γ-H2AX, as well as an elevated secretion of ROS. Co-treatment with TQ and PTX acted as senotherapeutic agents by enhancing apoptotic cell death through modulation of Bax/Bcl2/cleaved caspase-3 and β-galactosidase activity, and by increasing oxidative stress. Notably, the combination of TQ and PTX was shown to support a greater senotherapeutic effect. Conclusion and implications: Findings indicated that TQ and PTX acted as senotherapeutic agents in 5-FU-induced senescent CRC spheroids and may have potential as adjuvants to enhance CRC therapy.