Chitosan Nanoparticles Encapsulating Ipomoea digitata L. Tuber and Jatropha gossypiifolia L. leaf Extract Suppress NF-κB, Wnt/β-catenin, and MAPK Signaling Pathways in HCT116 and SW480 Colorectal Cancer Cells

Ipomoea digitata and Jatropha gossypiifolia are traditional medicinal plants that have been widely used in indigenous medicine for their anti-inflammatory and anticancer benefits. Despite their long-standing use, scientific understanding of how effective they are against colorectal cancer and how they might influence the cancer-related signaling pathways remains limited and requires further research. The study was aimed at evaluating the anticancer and anti-inflammatory properties of I. digitata tuber and J. gossypiifolia leaf extracts encapsulated in chitosan nanoparticles (CSNPs), particularly examining their effects on inhibiting NF-κB, Wnt/β-catenin, and MAPK signaling pathways in colorectal cancer models. Methanol extracts from the tubers of I. digitata and the leaves of J. gossypiifolia were encapsulated into chitosan nanoparticles through the ionic gelation method. The physicochemical characteristics of these chitosan nanoparticles, including particle size, zeta potential, and encapsulation efficiency, were analyzed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Their cytotoxic effects were evaluated against HCT116 and SW480 colorectal cancer cell lines using the MTT assay. Apoptosis induction was confirmed by observing increased caspase-3 activity and through fluorescence microscopy with an Annexin V/PI apoptosis detection kit. The expression of key oncogenic signaling molecules- NF-κB p65, β-catenin, ERK1/2, and p38 MAPK were examined by Western blotting and quantitative PCR. Additionally, anti-inflammatory effects were assessed by measuring levels of TNF-α, IL-6, and COX-2 using enzyme-linked immunosorbent assay (ELISA). The chitosan nanoparticles (CSNPs) exhibited a uniform size distribution with an average particle size of approximately 180 nm, and the encapsulation efficiency of the plant extracts was high (>85%). The nanoparticles demonstrated controlled release profiles over 48 hours. Treatment with extract-loaded CSNPs resulted in a significant reduction in cell viability in both HCT116 and SW480 colorectal cancer cells, with IC?? values of 22.3 ± 2.1 µg/mL and 34.8 ± 3.4 µg/mL, respectively, compared to free extracts (IC?? values: 40.5 ± 4.3 µg/mL for HCT116 and 52.1 ± 3.9 µg/mL for SW480). Apoptotic cell death was confirmed by increased caspase-3 activity, with a 2.3-fold increase in both cell lines following treatment with the extract-loaded CSNPs (p < 0.05). Fluorescence microscopy analysis of Annexin V/PI-stained cells revealed a significant increase in early and late apoptotic cells, with 43.5% and 35.6% of HCT116 and SW480 cells, respectively, exhibiting apoptosis after treatment, compared to 18.2% and 14.7% in the untreated control groups. Molecular analysis further revealed significant downregulation of key oncogenic proteins, including NF-κB p65, β-catenin, and ERK1/2. Additionally, the extract-loaded CSNPs significantly reduced the expression of pro-inflammatory cytokines such as TNF-α (by 45.3 ± 4.2%), IL-6 (by 48.7 ± 5.1%), and COX-2 (by 50.2 ± 3.8%) in treated cells. Chitosan nanoparticles carrying extracts from I. digitata and J. gossypiifolia have been shown to effectively slow down the growth of colorectal cancer cells by targeting important cancer-related and inflammatory pathways. This study not only validates the ethnomedicinal uses of these plants but also highlights their potential as promising natural options for developing new treatments against colorectal cancer.