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Synergistic Cytotoxicity between Elephantopus scaber and Tamoxifen on MCF-7-Derived Multicellular Tumor Spheroid

ABSTRACT: Elephantopus scaber Linn, a traditional herb, exhibited anticancer properties, and it was cytotoxic against the monolayer estrogen receptor-positive breast cancer cell line, MCF-7, in the previous study. In order to determine the potential of E. scaber as a complementary medicine for breast cancer, this study aimed to evaluate the synergism between E. scaber and tamoxifen in cytotoxicity using MCF-7 in the form of 3-dimensional multicellular tumor spheroid (MCTS) cultures. MCTS represents a more reliable model for studying drug penetration as compared to monolayer cells due to its greater resemblance to solid tumor. Combination of E. scaber ethanol extract and tamoxifen, which were used in concentrations lower than their respective IC50 values, had successfully induced apoptosis on MCTS in this study. The combinatorial treatment showed >58% increase of lactate dehydrogenase release in cell media, cell cycle arrest at the S phase, and 1.3 fold increase in depolarization of mitochondrial membrane potential. The treated MCTS also experienced DNA fragmentation; this had been quantified by TUNEL-positive assay, which showed >64% increase in DNA damaged cells. Higher externalization of phospatidylserine and distorted and disintegrated spheroids stained by acridine orange/propidium iodide showed that the cell death was mainly due to apoptosis. Further exploration showed that the combinatorial treatment elevated caspases-8 and 9 activities involving both extrinsic and intrinsic pathways of apoptosis. The treatment also upregulated the expression of proapoptotic gene HSP 105 and downregulated the expression of prosurvival genes such as c-Jun, ICAM1, and VEGF. In conclusion, these results suggested that the coupling of E. scaber to low concentration of tamoxifen showed synergism in cytotoxicity and reducing drug resistance in estrogen receptor-positive breast cancer.

SUBMITTER: Ho W

PROVIDER: S-EPMC8548115 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.

Project description:Breast cancer is one of the most common cancers among females and is associated with high mortality and morbidity rates. Several studies have demonstrated that combination treatments with natural products and tamoxifen can improve the sensitivity and cytotoxicity of oestrogen-positive breast cancer cells in response to tamoxifen. Celastrol, a triterpene from traditional Chinese medicine, has been proven to exert significant anticancer effects on various cancers. Our study is aimed at exploring the interactive antitumour effects of celastrol combined with tamoxifen and the potential underlying anticancer mechanisms in MCF-7 cells. The results from MTT assays, isobolographic analyses, and clonogenic cell survival assays revealed that a combination of celastrol and tamoxifen exerted synergistic cytotoxic effects in MCF-7 cells. The results from Annexin V/PI staining and flow cytometry analysis suggested that celastrol enhanced tamoxifen-mediated apoptosis. In addition, exposure to a combination of celastrol and tamoxifen inhibited cell proliferation by causing G1 phase cell cycle arrest. Moreover, the distribution of LC3 was monitored by immunofluorescence, and the changes in the LC3II and P62 levels detected by western blot analysis suggested that celastrol in combination with tamoxifen triggered autophagy. Furthermore, the decrease in p-Akt and p-mTOR in MCF-7 cells, along with the increase in the autophagy marker proteins LC3II and P62, suggested that the Akt/mTOR pathway might be involved in the triggering of cell autophagy by the combination treatment. However, in an MCF-7-implanted nude mouse model, it was possible to detect significantly decreased tumour volumes and tumour weights and decreased p-Akt and p-mTOR protein expression in the celastrol+tamoxifen group. Therefore, our study provides the first evidence that celastrol combined with tamoxifen exerts synergistic anticancer effects by inducing apoptosis and autophagy in MCF-7 cells. Considering the urgent need for novel therapeutic strategies in anticancer therapy, this combinatorial approach is worthy of further investigation.

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Synergistic Cytotoxicity between Elephantopus scaber and Tamoxifen on MCF-7-Derived Multicellular Tumor Spheroid

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