Effect of Aqueous and Methanol Extracts of Tradescantia zebrina and fluminensis on Human Cells Ashley Brauner, Michael Pickart, Ph,D., University of Wisconsin-Stout Collaborating Students: Benjamin Leist, Danielle Moehring, Calli Walsh, Samantha Langsford, and Lexi Blise Tradescantia zebrina and Tradescantia fluminensis have medicinal properties. Extracts of these plants may have anticancer characteristics. Assays were preformed that measured doubling time and clonogenic survival of SCC-13y (squamous cell carcinoma), HFF-1 (human foreskin fibroblasts), and A549 (lung adenocarcinoma) cells. Proliferation inhibition was determined via growth curve analysis. Compared to the negative control of sterile water, cancer cell proliferation was decreased with the addition of T. zebrina treatments. This study confirms the general inhibitory effects of T. zebrina and T. fluminensis extracts on cancerous and non-cancerous cells, allowing further research to be conducted involving different cell lines and methods of extraction. T. zebrina on HFF-1 Cells T. zebrina on HFF-1 600000 700000 Negative Control 600000 B 500000 1:40 Tz Extract 500000 Total Cell Count A 400000 Total Cell Count 400000 Expon. (Negative Control) y = -7500x + 71500 300000 Linear (1:40 Tz Extract) 200000 300000 0 Hours 72 Hours 200000 y = 24119e1.0817x 100000 100000 0 24 48 72 0 Negative Control Time (Hours) C T. zebrina on SCC-13y Cells 1:40 Tz Extract T. zebrina on SCC-13y Cells 2,500,000 D 2,500,000 Negative Control Total Cell Count 2,000,000 2,000,000 1:40 Tz Decoction 1,500,000 1,000,000 Expon. (Negative Control) Total Cell Count Abstract Summary Preliminary Investigation: Inhibition of Proliferation Extract Treatment Procedure 1,500,000 0 Hours 94 Hours 1,000,000 y = 132445e0.7075x 500,000 Linear (1:40 Tz Decoction) 500,000 y = -33500x + 292500 0 0 48 72 0 94 T. fluminensis on SCC-13y Cells 1:40 Tz Decoction E Negative Control T. fluminensis on SCC-13y Cells 1,600,000 F 1,400,000 1,400,000 Cell Count (1:40 Tf Decoction) 1,200,000 Figure 1. A) T. fluminensis plant B) T. zebrina plant Extraction Methods Table 1. Comparison between methanol and aqueous extraction methods, along with challenges faced during development and the solutions implemented to solve the problems. Benefits Aqueous Extraction Methanol Extraction Simple Procedure Clear Extract Sterile Definite Concentration Preserves Molecules Challenges Indefinite Concentrations Solutions Clonogenic Survival Methods 1,200,000 Negative Control 1,000,000 1,000,000 Linear (Cell Count (1:40 Tf Decoction)) 800,000 y = 35783e0.0255x 600,000 Expon. (Negative Control) 400,000 y = 783.85x + 88875 Total Cell Count Total Cell Count Figure 2 (above). Flowchart of treatment procedure. Aqueous extracts of Tradescantia zebrina and Tradescantia fluminensis were diluted and applied to SCC-13y cells. Future experiments allow for the use of different concentrations of T. fluminensis and T. zebrina methanol extract applied to different cell lines. Figure 3 (below). Clonogenic survival assays were performed using confluent SCC-13y plates of a 2.5% aqueous treatment and a control of sterile water. Each confluent plate was trypsinized and counted using a hemacytometer. Cells were seeded in triplicate for both the experimental and control groups at 100 and 200 cell densities in p60 dishes in DMEM media supplemented in 10% FBS and 1% P/S. Cells were allowed to colonize for one week and were then fixed with 4% paraformaldehyde and treated with crystal violet stain. Stained cells were then counted to determine clonogenic survival rate. 800,000 0 Hours 144 Hours 600,000 400,000 200,000 200,000 0 0 0 50 100 150 200 1:40 Tf Extract Negative Control Time (Hours) Figure 4. A) Growth curve analysis of a 2.5% T. zebrina aqueous extract. ~62,500 HFF1 cells were seeded, treated with T. zebrina aqueous extract, and counted 24 and 48 hours post-seeding. B) Comparative analysis of 2.5% T. zebrina extracts on HFF1 cells as compared to the negative control of sterile water. C) Growth curve analysis of 2.5% T. zebrina extracts on SCC-13y cells. ~280,000 SCC-13y cells were plated in p60 dishes and treated with 2.5% T. zebrina aqueous extracts using equal concentrations of sterile water as a negative control. Cells were harvested and counted at 24, 72, and 96 hours post-seeding D) Comparative analysis of 2.5% T. zebrina extracts with the negative control of sterile water on SCC-13y cells 0 hours post-seeding and 94 hours post-seeding. E) Growth curve analysis of 2.5% T. fluminensis extracts on SCC-13y cells. ~31,250 SCC-13y cells were plated and treated with 2.5% T. fluminensis aqueous extract using sterile water as a negative control. Control and treatment plates were harvested and counted with a hemacytometer after 48, 96, and 144 hours post-seeding in order to generate a growth curve F) Comparative analysis of 2.5% T. fluminensis extracts with the negative control of sterile water 0 hrs post-seeding and 144 hours post-seeding. Error bars are not shown for all graphs due to a lack of triplicate testing. Future Goals and Research Complex Procedure Murky Extract Non-sterile More Concentrated Extracts Used Improved Methanol Used 0.22-µm Filter Extraction Method Sterilizer Used Centrifuge Evaporator 1. Clonogenic Survival Assay – to determine the survival rate of cells after the removal of T. zebrina and fluminensis extract treatment. 2. Zebrafish Toxicity Treatment – to determine if the antiproliferative properties of the extract are due to cytostatic activity within the cell or due to toxicity of the extract. 3. Flow cytometry readings using fluorescence-activated cell sorter of cells treated with T. zebrina or T. fluminensis extract would generate a cell cycle index that could reveal cytostatic activity within the cell.