The Use of Shikonin to Reduce Viability of Breast Cancer Cell Lines T47D and MDA-MB-231 - PowerPoint Presentation

Slide1

The Use of

Shikonin

to Reduce Viability of Breast Cancer Cell Lines T47D and MDA-MB-231

Alexis Clark

1

and Rebecca Haberman

1

1

Dept. of Biology, Mary Baldwin University, Staunton, VA

Introduction

In the United States alone 1 in 8 women will develop invasive breast cancer over the course of their lifetime(1).Of these invasive breast cancers triple-negative breast cancer will account for around 15% of all breast cancers (2).Estrogen receptor positive (ER+) breast cancers express receptors that are confer sensitivity to commonly used treatments.Triple-negative breast cancer that lacks all of the receptors that are commonly found in other types of breast cancer. Natural compounds are being investigated as potential treatments that will improve the outcomes studied of triple-negative breast cancer. Shikonin is a naturally occurring drug that is being investigated for its cytotoxic effects in various cancer cell lines including liver and lung.Breast cancer cell lines T47D and MDA-MB-231 are both human breast cancer cell lines that are ER+ and triple-negative.

Materials and Methods

Cell Culture T47D cells gown in RPMI-1640 and MDA-MB-231 cells grown in DMEM media80% confluence cells treated with concentrations 0.02µg/mL, 0.1µg/mL, 0.5µg/mL, and 2.5µg/mL. Grown at time points 2, 6, and 24 hoursTrypan Blue Exclusion Detached cells from 6 well plate Mixed 20µL cells and 20µL of Trypan blue solution Counted live and dead cells using hemocytometerCaspase-3 AssayT47D and MDA-MB-231 cells used at concentrations 0.1µg/mL and 0.5µg/mL at time point 6 hoursPositive control was camptothecin at 6 and 24 hoursProtein extraction and absorbances read at 750nmCaspase-3 solutions mixed with cell line samples and incubated for 2 hours Absorbances read at 405nm and recorded

Results

Results

MDA-MB-231 Decrease in cell viability detected within the various concentrations of SHK.Showed significance at concentrations 0.5µg/mL and 2.5µg/mLCondition determinate factor no time interaction or condition and time interaction.SHK reduces viability by dose-dependence in MDA-MB-231 cell line.

Figure 4. Percentage of Live MDA-MB-231 cells compared to control. Bars indicate standard error calculated using Excel. 2-way Anova performed for statistical analysis showed significance (F(14,59)=33.4;p<0.001; Condition= (F(4,59)=111.6;p<0.001); Condition*Time= (F(8,59)=2.19;p<0.05)

Figure 5.

Pictures obtained at time point 24 hours in MDA-MB-231 cells with varying concentrations of

Shikonin.

Figure 6.

MDA-MB-231 average cell death across all time points. 2-way Anova performed for statistical analysis showed significance (*** indicates significance (F(14,59)=20.9;P<0.001; Condition= (F(4,59)=71.1;p<0.001)

Conclusion

T47DShikonin treatment decreases number of viable cellsIncreases number of cell death SHK shows time and dose-dependent responsePotential treatment for ER+ breast cancersMDA-MB-231Shikonin treatment decreases number of viable cellsIncreases amount of cell deathSHK shows dose-dependent response in MDA-MB-231 cancer cell linePotential treatment for triple-negative breast cancers

Future Research

Shikonin mechanism is still unknown. Future research can be aimed at determining how Shikonin introduces these various effects within these cancer cell lines. Research in other cancer studies shows Shikonin acts through mitochondrial mechanisms.

Acknowledgements

I am grateful to Dr. Rebecca Haberman and the Department of Biology for the extensive support throughout this research project. I would also like to acknowledge fellow classmates and our lab coordinator Megan Alderman for the support and assistance within the project.

References

American Cancer Society. Types of breast cancer: Different Breast Cancer Types. (n.d.). Retrieved October 21, 2021, from https://www.cancer.org/cancer/breast-cancer/about/types-of-breast-cancer.html. American Cancer Society. (2021, November 12). Triple-negative Breast Cancer: Details, Diagnosis, and Signs. American Cancer Society. Retrieved February 18, 2022, from https://www.cancer.org/cancer/breast-cancer/about/types-of-breast-cancer/triple-negative.html#written_by Medina, M. A., Oza, G., Sharma, A., Arriaga, L. G., Hernández Hernández, J. M., Rotello, V. M., & Ramirez, J. T. (2020). Triple-Negative Breast Cancer: A Review of Conventional and Advanced Therapeutic Strategies. International Journal of Environmental Research and Public Health, 17(6), 2078. https://doi.org/10.3390/ijerph17062078Lu, L., Qin, A., Huang, H., Zhou, P., Zhang, C., Liu, N., Li, S., Wen, G., Zhang, C., Dong, W., Wang, X., Dou, Q. P., & Liu, J. (2011). Shikonin Extracted from Medicinal Chinese Herbs Exerts Anti-Inflammatory Effect via Proteasome Inhibition. European Journal of Pharmacology, 658(2-3), 242–247. https://doi.org/10.1016/j.ejphar.2011.02.043Wang, H., Liu, Z., Li, X., Zhao, R., Pu, Y., Wu, H., & Guan, W. (2018). Shikonin causes apoptosis by disrupting intracellular calcium homeostasis and mitochondrial function in human hepatoma cells. Experimental and Therapeutic Medicine, 15(2), 1484–1492. https://doi.org/10.3892/etm.2017.5591

T47D Decrease in cell viability detected within the various concentrations of SHKShowed significance at concentrations 0.02µg/mL, 0.1µg/mL, 0.5µg/mL, and 2.5µg/mLCondition, time, and condition and time interactions significant SHK reduces viability by time and dose-dependence in T47D cell line

Figure 1.

Percentage of Live T47D cells compared to control. Bars indicate standard error calculated using Excel. 2-way Anova performed for statistical analysis showed significance (F(14,59)=51.9;p<0.001; Time= (2,59)=35.2;p<0.001; Condition= (F(4,59)=147.8;p<0.001; Condition*Time= (F(8,59)=8.2;p<0.001)

Figure 2.

Pictures obtained at time point 24 hours in T47D cells with varying concentrations of Shikonin.

Figure 3.

T47D average cell death across all time points. 2-way

Anova performed for statistical analysis showed significance. (F(14,59)=33.2,p<0.001; Condition=(F(4,59)=109.3;p<0.001); Time=(F(2,59)=6.886;p<0.05)