Hopea Odorata Extract Can Efficiently Kill Breast Cancer Cells and Cancer Stem-Like Cells in Three-Dimensional Culture More Than in Monolayer Cell Culture

Introduction: The breast cancer cells with CD44⁺CD24^- phenotype are known to play an important role in tumorigenesis, drug resistance, and cancer recurrence. Breast cancer cells with CD44⁺CD24^- phenotype are cultured in three-dimensional (3D) stereotype showing the recapitulation of tumors in vivo such as cell differentiation, heterogeneity, and microenvironment. Using this 3D model in anti-cancer compound research results in a more accurate reflection than conventional monolayer cell culture. This study aimed to identify the antitumor activity of Hopea odorata methanol extract (HO-MeOH-E) on breast cancer cells and cancer stem-like cells in both models of three-dimensional culture (3D) and monolayer cell culture (2D).

Methods: HO-MeOH-E was produced from Hopea odorata plant. The VN9 breast cancer cells (VN9) were collected and expanded from the previous study. The breast cancer stem-like cells (VN9CSC) were sorted from the VN9 based on phenotype CD44⁺CD24^-. Both VN9 and VN9CSC were used to culture in monolayer culture (2D) and organoids (3D) before they were used to treat with HO-MeOH-E. Two other anticancer drugs, doxorubicin and tirapazamine, were used as references. The antitumor activities of extracts and drugs were determined via two assays: antiproliferation using the Alamar blue assay and cell cycle assay.

Results: The results showed that HO-MeOH-E was sensitive to both VN9 and VN9CSC in 3D more than 2D culture (IC₅₀ on 3D organoids 144.8 ± 2.172 μg/mL and on 2D 340.2 ± 17.01 μg/mL for VN9CSC (p < 0.001); IC₅₀ on 3D organoids 2055 ± 82.2 μg/mL and on 2D 430.6 ± 8.612 μg/mL for VN9 (p < 0.0001), respectively). HO-MeOH-E inhibits VN9CSC proliferation by blocking S phase and increasing the populations of apoptotic cells; this is consensus to the effect of tirapazamine (TPZ) which is used in hypoxia-activated chemotherapy.

Conclusion: Taken these results, HO-MeOH-E has the potential effect in hypoxia-activated chemotherapy specifically on breast cancer stem-like cells with CD44⁺CD24^- phenotype.

Keywords: 3D cultured; Breast cancer; Breast cancer stem cell; Hopea odorata; Hypoxia; Organoids.