The Effects of Vitamin C on MG-63 Cancer Cell Survivorship Joe Ziccarelli Grade 12 Central Catholic High School PJAS 2015

An Overview of Cancer Cells Cancer cells are cells that grow and divide at an irregular, unregulated pace. Apoptosis does not occur in cancerous cells; their mutations are passed on to the second generation, eventually clustering and forming tumors. Tumors can be malignant (aggressive) or benign.

MG63 Cancer Cell Line Human cancer cell line Osteosarcoma cells, an aggressive form of bone cancer Useful model to test the effects of variables on cancer cell proliferation

Antioxidants Molecules capable of slowing or preventing the oxidation of other molecules May be able to prevent cancer and coronary heart disease Body produces antioxidants Can obtain through Diet Past studies have shown that the antioxidant nature of Vitamin C has anti-tumoregenic effects

Variable: Ascorbic Acid (Vitamin C) Antioxidant Enzyme cofactor In Oranges, Strawberries, and Grapefruit Recommended daily intake: 60 mg The disease scurvy occurs from lack of Vitamin C

Purpose To determine the effects of ascorbic acid on MG-63 cell survivorship.

Hypotheses Null Hypothesis: Ascorbic will not have a significant effect on MG63 cell survivorship. Alternative Hypothesis: Ascorbic Acid will significantly alter the survivorship of MG63 cells.

Materials Cryotank Four 75mm2 tissue culture treated flasks, Sixteen 25 mm2 tissue culture treated flasks MG63 Osteosarcoma Cancer Cell Line Trypsin-EDTA DMEM Media 1 Molar Ascorbic Acid Solution Incubator Evo Imaging System Macropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips Labeling Tape Hemacytometer Sterile PBS Ethanol (70% and 100%) Laminar Flow Hood UV and Sterilizing Lamp Purple Nitrile gloves Safety goggles

Procedure 1: Cell Culturing A 1 mL aliquot of MG63 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in one 75mm2 culture flask yielding a cell density of approximately 106 to 2 x 106 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 4 x 106 to 5 x 106 cells/mL was reached. The culture was passed into two sets of 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

Procedure 1: Proliferation Experiment- Day 0 (Addition of Variable) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL). 0.1 mL of the cell suspension was added to eight 25 mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells per flask. The stock solution of ascorbic acid (one molar) was created using 50 mL of ethanol and 10 g of ascorbic acid. This was then used in a serial dilution to create a 10-2, 10-4, 10-6 M concentration. The cells were incubated at 37°C, 5% CO2 for the remainder of the study.

Procedure 2: Cell Culturing A 1 mL aliquot of MG63 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in one 75mm2 culture flask yielding a cell density of approximately 106 to 2 x 106 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 4 x 106 to 5 x 106 cells/flask was reached. The culture was passed into two sets of 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

Procedure 2: Proliferation Experiment- Day 0 (Addition of Variable) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL). 0.1 mL of the cell suspension was added to eight 25 mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells per flask. The stock solution of ascorbic acid (one molar) was created using 50 mL of ethanol and 10 g of ascorbic acid. This was then used in a serial dilution to create a 10-2, 10-4, 10-6 M concentration. The cells were incubated at 37°C, 5% CO2 for the remainder of the study.

Procedure: Proliferation Experiment- Days 1 and 3 Using one flask from each group, cell densities were determined as follows: The cells were trypsinized and collected into cell suspension. 25 µl aliquots were transferred to a Hemocytometer for quantification (eight counts per flask). Day 1 and Day 3 The previous procedure for determining densities was used again, and a Evo Imaging System was used to take images of representative areas of each flask.

Results of Proliferation Analysis (MG63) P-value 4.15E-15 P-value 7.25E-22

Dunnett’s Test (Vitamin C Exposure After Two Days) Concentration T-Value T-Critical (0.05) Variation MG63 - 10^-6 M 2.057 2.88 Insignificant 10^-4 M 18.322 Significant 10^-2 M 26.357

Results of Proliferation Analysis (MG63) P-value 2.09E-13 P-value 2.15E-13

Dunnett’s Test (Variable Added Without Two Day Proliferation) Concentration T-Value T-Critical (0.05) Variation MG63 - 10^-6 M 0.095 2.88 Insignificant 10^-4 M 19.205 Significant 10^-2 M 26.551

Conclusions MG63 (variable added after two day proliferation) Based upon the results gathered from the ANOVA and Dunnett’s statistical analyses, it appears that the addition of ascorbic acid at higher concentrations significantly affects cancer cell proliferation. Therefore, the null hypothesis can be rejected. MG63 (variable added without two day proliferation)

Future Changes Limitations Extensions Hemacytometer counts in proliferation experiment were subject to clumping error Limited range of concentrations Limited days of exposure Only accessed proliferation/survivorship Include further trials in order to maximize data accuracy Use a wider range of concentrations Test synergistic effects of other vitamins

Acknowledgements Mark Krotec, PTEI Merisant Company

Works Cited http://cancerres.aacrjournals.org/content/ 40/3/734.full.pdf http://www.cancer.gov/cancertopics/pdq/c am/highdosevitaminc/patient/page2 http://www.mayoclinic.org/diseases- conditions/cancer/expert- answers/alternative-cancer-treatment/faq- 20057968

Works Cited (cont.) http://lpi.oregonstate.edu/s- s00/vitaminc.html http://www.webmd.com/cancer/news/201 40205/intravenous-vitamin-c-may-boost- chemos-cancer-fighting-power

Statistical Analyses of the Proliferation Results ANOVA Compares variation within groups to variation between groups. Using the ANOVA, a p-value less than the alpha of .05 was gathered (significant variation). Reject the null hypothesis. Dunnett’s test Compares each experimental group to control individually. 0.05 alpha was used, and the t-value compared to the t- critical value of 2.88

Additional Information DMEM Serum - 1% and Complete 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])