Agarose-Based 3D Spheroid Model to Evaluate the Anticancer Activity of the Curcumin Analog CCA-1.1 in Triple-Negative...

Three-dimensional (3D) cell culture systems provide superior simulation of the tumour microenvironment compared to traditional two-dimensional (2D) cultures. Chemoprevention agent curcumin analog-1.1 (CCA-1.1), a synthetic curcumin derivative, has demonstrated promising anticancer properties against triple-negative breast cancer (TNBC). This study aimed to develop and use a 3D agarose-based culture system for MDA-MB-231 cells to comprehensively evaluate the efficacy of CCA-1.1 as an anticancer agent.

MDA-MB-231 breast cancer cells were cultured in both a 2D monolayer and a 3D agarose-based spheroid system. Cytotoxicity was assessed using CCK-8 assays, and IC50 values were determined. Spheroid formation capacity was evaluated and compared with that of doxorubicin. Propidium iodide staining was performed to assess cell viability and structural integrity. Gene expression analysis of epithelial-mesenchymal transition (EMT) markers and metastasis-related proteins (E-cadherin, MMP2, and MMP9) was conducted using qRT-PCR.

CCA-1.1 demonstrated IC50 values of 1.48±0.28 µM in 2D cultures versus 6.12±0.27 µM in 3D cultures, while doxorubicin showed IC50 values of 0.79±0.22 µM (2D) and 2.65±0.3 µM (3D). CCA-1.1 reduced spheroid formation by 40% compared to controls, whereas doxorubicin achieved 96% inhibition. Propidium iodide staining showed that CCA-1.1 induced cell death while preserving the spheroid structure, unlike doxorubicin, which disrupted the architecture. Expression level analysis revealed that CCA-1.1 reduced MMP2 (0.15-fold, p=0.0001) and MMP9 (0.25-fold, p<0.0001) levels while increasing E-cadherin levels by 1.54-fold (p=0.379).

Our study using a 3D agarose-based spheroid culture confirms the cytotoxicity of CCA-1.1 against MDA-MB-231 and showed that the compound inhibited spheroid formation and epithelial-mesenchymal transition, and reduced the expression levels of matrix metalloproteinases. These data support the development of CCA-1.1 as a candidate chemotherapeutic agent for TNBC.