Student Instituto Tecnológico y de Estudios Superiores de Monterrey Guadalupe, United States
Introduction: Breast cancer is the second leading cause of death for women worldwide. In 2022, 2.3 million women were diagnosed, leading to 670,000 deaths. Common treatments, including surgery, chemotherapy, and radiotherapy, often have severe side effects that significantly decrease the quality of a patient's life.
Hibiscus sabdariffa (Hs) is a flowery plant commonly used in culinary applications and contains polyphenols and flavonoids. These compounds are important because they protect against chronic diseases, including type 2 diabetes, cardiovascular diseases, obesity, and cancer.
Polyphenols have shown anticancer effects such as interference with redox balance, cell cycle arrest, pro-apoptotic activity, autophagy activation, and anti-inflammatory effects, among others. Specifically, the effect of Hs on 2D cultures of the breast cancer cell line MCF-7 has been studied and found to reduce cell viability, cell migration, and cell invasion, and to induce apoptosis in a dose-dependent manner. Further research in 3D cultures could help better comprehend these effects in a real-life scenario since they better mimic tumor behavior.
Because of this, our aim was to evaluate the effect of Hibiscus sabdariffa extract (HsE) on MCF-7 spheroids using different concentrations of the plant’s ethanolic extract.
Materials and
Methods: We used the MCF-7 cell line to create 100 µm wide spheroids of 2 million cells each, using agarose gel molds. We obtained the HsE through maceration of the dried plant and a 40% ethanolic extraction (1:5 ratio). Then, we exposed the spheroids to 3 different concentrations of HsE diluted in the medium: low (8 mg/mL), medium (16 mg/mL), and high (32 mg/mL). Control groups included a negative control with only culture media and spheroids, and a positive control treated with doxorubicin (4μM). Spheroids were exposed to these concentrations for 8 days.
We assessed spheroid morphology and cell viability using brightfield microscopy with an inverted microscope, a Live/Dead assay with the Invitrogen Live/Dead Cell Imaging Kit, and a glucometry assay with the Accu-Chek Active glucometer and test strips.
For brightfield microscopy, our aim was to study the morphological changes in the spheroids over time; we analyzed the images in ImageJ software. For the Live/Dead assay, our purpose was to study the quantity of live or dead cells within the spheroids; we also analyzed these images in ImageJ to obtain viability percentages. Finally, we used the glucometry assay to indirectly measure the metabolic activity of the spheroids; we used the glucometer and test strips as usual, and the sample was a 10 μL drop of culture media.
Results, Conclusions, and Discussions: Our results showed that HsE affected the development of MCF-7 spheroids, with a reduction in cell viability inversely proportional to the extract concentration and to time.
At medium and high concentrations, cell viability significantly decreased from approximately 98% on day 0 to about 2% by day 5, with no further significant change observed between day 5 and day 8. A similar trend was observed in the positive control (doxorubicin), where viability decreased significantly from day 0 to day 5 and then remained stable until day 8. Conversely, at low concentration, cell viability decreased significantly from around 98% on day 0 to approximately 30% on day 5, and further reduced to about 10% by day 8. These results demonstrate that while the low concentration had a slower effect, medium and high concentrations had a more rapid and equal impact.
According to the literature, in 2D cultures, there is a threshold HsE concentration (~0.5 mg/mL) above which the effect plateaus. We can infer that in 3D cultures, this threshold lies between the low and medium concentrations tested.
Additionally, we observed a significant reduction in glucose consumption at the low concentration from day 1 to day 7, while medium and high concentrations showed no consumption on any day, thus implying that these spheroids were not metabolically active and dying. We observed a significant increase in glucose consumption in the negative control group that correlated with the morphology results (significantly larger spheroid area at day 8 compared to the rest of the groups). Moreover, the negative control maintained consistent viability of around 98% throughout the experiment. Thus, we can infer that the experimental and positive control groups did not grow or develop.
In conclusion, HsE demonstrates a decreasing effect on cell viability in MCF-7 spheroids, with medium and high concentrations acting rapidly, while the low concentration exerts a more gradual effect. While optimization of dosing still needs to be determined, these findings suggest the potential of HsE in breast cancer treatment.
Acknowledgements (Optional): We acknowledge MSc. Sara Pedroza for her technical advice and guidance through the project. Also, the Álvarez-Trujillo Lab and the Tecnológico de Monterrey for providing access to their facilities.
