The EFFECTS OF OILS ON MG63 CANCER CELLS Carolyn Guzikowski Oakland Catholic High School ‘20

An Overview of Cancer Cells Cells that grow and divide at an irregular and unregulated pace Apoptosis (cells programmed death) stops in cancerous cells; their mutations are passed on to the second generation, and eventually cluster and form tumors Tumors can either be malignant (aggressive) or benign Photo Credits: sites.duke.edu

MG63 Cell Line Osteosarcoma cells, an aggressive form of bone cancer Useful model to test the effects of variables on cancer cell proliferation Photo Credits: neutron.ujf.cas.cz

2 Variables: Essential Oils Turmeric Antibiotic Cancer preventative Anti-tumor Antioxidant Aids in fighting colon cancer Ginger Supports healthy digestion Treats nausea Helps in occasional joint or muscle discomfort Photo Credits: up-nature.com and shape.com

Purpose To examine the effects of Ginger Oil and Turmeric Oil on MG63 Cell count proliferation and survivorship.

Hypothesis Null Hypothesis Alternate Hypothesis The different concentrations of Ginger Oil and Turmeric Oil will not affect the cell count proliferation and survivorship. The different concentrations of Ginger Oil and Turmeric Oil will affect the cell count proliferation and survivorship.

Materials Cryotank DMEM Media (4 mM L- glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) One 75mm2 tissue culture treated flasks Hemocytometer Permanent marker Incubator Six 25 mm2 tissue culture treated flasks 24 well plate Aspirating Vacuum Line Test tube rack 10% fetal bovine serum Nikon Inverted Compound Optical Scope Sterile Filter MG63 Osteosarcoma Cancer Cell Line Laminar Flow Hood Trypsin Laminar Flow Hood UV Sterilizing Lamp Dye reagent concentrate Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL) Spectrophotometer Labeling Tape 100% Pure Turmeric Oil Sterile PBS Micropipettes + sterile tips 100% Pure Ginger Oil Ethanol (70%) Distilled water Nikon Inverted Microscope

Combined Variable O (Control) L (Low) H (High) OO OL OH LO LL LH HO HL Back = Ginger C = 0 mL Red = Turmeric L = .005 mL (5 µl) H = .05 mL (50 µl)

Procedure: Cell Culturing A 1 mL aliquot of MG63 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75cm2 culture flask yielding a cell density of approximately 106 to 2x106 cells The media was replaced with 15 mL of fresh media to remove cryo- freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL was reached The culture was passed into 18 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2

Procedure: Proliferation Experiment Addition of Variable Cells from a T75 flask were resuspended after trypsinization to a density of approximately 300-500K/mL 4 mL of 10% DMEM media was added to each T25 flask 0.5 mL of cell suspension was transferred to 12 T25 flasks. Flasks were placed back into incubator and cells were allowed to attach for several hours 0.1 mL of Ginger oil was added to 9.9 mL distilled water to create a stock of 1% Serial dilution to create a 0.01% (stock B) was then created with .1 mL of stock A into 9.9 mL of 10% DMEM. All were sterile filtered using a 0.22 micron filter Stock solution process repeated for Low and High Turmeric concentrations T25 flasks were removed from incubator and the variable was added to reach desired concentrations

Variable Concentrations Proliferation Experiment Con Con/Low Low/Con Low/Low Con/High Cells 0.5 mL Media 4.5 mL 4.495 mL 4.4 mL 4.45 mL Stock 0 mL .005 mL .01 mL .05 mL Total 5 mL

Variable Concentrations Cont’d High/Con High/High Low/High High/Low Cells 0.5 mL Media 4.45 mL 4.4 mL 4.445 mL Stock .05 mL .1 mL .055 mL Total 5 mL

Procedure: Cell Counting Day 1 and Day 2 The cells were trypsinized and collected into cell 2ml suspension. 10 µl aliquots were transferred to a Hemocytometer for quantification (eight total counts).

Statistical analysis of the proliferation results ANOVA (Single/Double Factor) Compares variation within groups to variation between groups Using the ANOVA, a P-value less than the alpha of .05 was gathered (significant variation) Allows the rejection of the null hypothesis

P-Value: 4.11E-16 Ginger

P-Value: 3.65E-20 Control Turmeric Ginger

P-Value: 6.26E-35 Control Ginger and Turmeric Lows

P-Value: 4.55E-35 Control Low Ginger and High Turmeric

Day 1 Counts: Control, Low, High

Day 1 Counts: Control Low High

Day 2 Counts:

Day 2 Counts:

D2 LH

Conclusions The null is rejected, Turmeric and Ginger appeared to have significant effects on MG63 Cell count proliferation and survivorship.

Future Changes Limitations Extensions Cell counts can vary Only one cell line used Use a wider range of concentrations Compare Turmeric and Ginger to Nigella Sativa (Black Cumin)

Works Cited Mark Krotec, PTEI Zheng, J., Zhou, Y., Li, Y., Xu, D.-P., Li, S., & Li, H.-B. (2016). Spices for Prevention and Treatment of Cancers. Nutrients, 8(8), 495. http://doi.org/10.3390/nu8080495 MG-63 (ATCC® CRL-1427™). (n.d.). Retrieved February 01, 2018, from https://www.atcc.org/Products/All/CRL- 1427.aspx#generalinformation