Cancer stem cells CSCs (tumour-initiating cells) are responsible for cancer metastasis and recurrence associated with resistance to conventional chemotherapy. This study generated MBA MD231 3D cancer stem cells enriched spheroids in serum-free conditions and evaluated the influence of combined doxorubicin/thymoquinone-loaded cockle-shell-derived aragonite calcium carbonate nanoparticles. Single loaded drugs and free drugs were also evaluated. WST assay, sphere forming assay, ALDH activity analysis, Surface marker of CD44 and CD24 expression, apoptosis with Annexin V-PI kit, cell cycle analysis, morphological changes using a phase contrast light microscope, scanning electron microscopy, invasion assay and migration assay were carried out; The combination therapy showed enhanced apoptosis, reduction in ALDH activity and expression of CD44 and CD24 surface maker, reduction in cellular migration and invasion, inhibition of 3D sphere formation when compared to the free drugs and the single drug-loaded nanoparticle. Scanning electron microscopy showed poor spheroid formation, cell membrane blebbing, presence of cell shrinkage, distortion in the spheroid architecture; and the results from this study showed that combined drug-loaded cockle-shell-derived aragonite calcium carbonate nanoparticles can efficiently destroy the breast CSCs compared to single drug-loaded nanoparticle and a simple mixture of doxorubicin and thymoquinone.
Background: Combination chemotherapy of anticancer drugs is extensively being researched since it could reduce multidrug resistance and side effects as a result of lower dosage of each drug. In this study, we evaluated the effects of doxorubicin-loaded (Dox-ACNP), thymoquinone-loaded (TQ-ACNP) and a combined doxorubicin/thymoquinone-loaded cockle shell-derived aragonite calcium carbonate nanoparticles (Dox/TQ-ACNP) on breast cancer cell line and compared with their free drugs counterpart. Methods: Cell viability using MTT assay, apoptosis with Annexin V-PI kit, morphological changes using contrast light microscope, scanning electron microscope and transmission electron microscope, cell cycle analysis, invasion assay, and scratch assay were carried out. The cell viability was evaluated in breast cancer cell line (MDA MB231), normal breast cells (MDF10A) and normal fibroblast (3T3). Results: MDA MB231 IC50 dosages of drug-loaded nanoparticle were not toxic to the normal cells. The combination therapy showed enhanced apoptosis, reduction in cellular migration and invasion when compared to the single drug-loaded nanoparticle and the free drugs. Scanning electron microscope showed presence of cell shrinkage, cell membrane blebbing, while transmission electron microscope showed nuclear fragmentation, disruption of cell membrane, apoptotic bodies, and disruption of mitochondrial cistern. Conclusion: The results from this study showed that the combined drug-loaded cockle shell-derived aragonite calcium carbonate nanoparticles (Dox/TQ-ACNP) showed higher efficacy in breast cancer cells at lower dose of doxorubicin and thymoquinone.