In Vitro evaluation of the therapeutic efficacy of a modified bioactive phytochemical structure in the HT-29 human colon adenocarcinoma model

Background and Aim: Colorectal cancer (CRC) ranks third globally in cancer incidence and fourth in cancer-related mortality, with its prevalence on the rise. Chemotherapeutic drugs' severe side effects and the emergence of multi-drug resistance hinder treatment efficacy. Hence, developing novel drug candidates from natural bioactive compounds capable of multi-pathway anti-carcinogenic effects with minimal toxicity is imperative. Our thesis aims to create precursor molecules for alternative anti-cancer drugs using bioactive phytochemicals like glucosamine, curcumin, and gallic acid. Material and Methods: Hybrid bioactive molecules SI-1 and GK-1, which are derivatives of phytochemicals curcumin, gallic acid, and glucosamine, were synthesized and structurally characterized using 1H-NMR, FTIR, and LC/MS analyses. The anti-colon cancer function of our molecules against oxaliplatin (OX), 5-fluorouracil (5-FU), and the combination therapy FOLFOX, commonly used in CRC treatment, was investigated using MTT assays with HT-29 cells. IC50 values were obtained using GraphPad Prism-8. Additionally, the synthesized chemicals were combined with OX and 5-FU, the components of FOLFOX, and MTT data were evaluated. The interactions within the combinations were interpreted using the Chou-Talalay method via CompuSyn Software. Results: Structural analysis confirmed SI-1 and GK-1 synthesis success, and MTT assays demonstrated their anti-colon cancer properties. IC50 values after 48 and 72 hours were as follows: SI-1, 333 µM and 56.65 µM; GK-1, 314.5 µM and 178.8 µM; 5-FU, 419.7 µM and 143.8 µM; OX, 26.11 µM and 16.76 µM, respectively. Our experiments also revealed that the combinations of SI-1 and GK-1 with 5-FU and OX inhibited the proliferation of HT-29 cells more effectively than the conventional therapeutic FOLFOX. CompuSyn analyses indicated that while the components of FOLFOX, 5-FU, and OX mostly exhibited an antagonistic relationship, the combinations of SI-1 and GK-1 with 5-FU and OX predominantly displayed synergistic interactions. Conclusion: Our products, SI-1 and GK-1, exhibited cytotoxic effects on in vitro human colon adenocarcinoma HT-29 cells, suppressing their proliferation. When our products were separately combined with the components of the conventional therapeutic FOLFOX, OX, and 5-FU, it was found that their abilities to suppress cancer cell proliferation were more effective than FOLFOX. Our experiments have highlighted the unique potentials of our original chemical structures, SI-1 and GK-1, as promising precursor molecules for human anti-colon cancer drugs. Keywords: Colon cancer, drug discovery, 5-fluorouracil, oxaliplatin, FOLFOX