Coenzyme Q-10 Effects on Mammalian Cell Behavior. Clayton Gentilcore Grade 11 Central Catholic High School PJAS 2013

Transcription

1 Coenzyme Q-10 Effects on Mammalian Cell Behavior Clayton Gentilcore Grade 11 Central Catholic High School PJAS 2013

2 An Overview of Stem Cells Unspecialized cells capable of renewing themselves through cell division Under certain physiological or experimental conditions, they can be induced to become tissue- or organspecific cells with special functions

3 C2C12 Stem Cell Line Subclone of the mus musculus (mouse) myoblast cell line Immortalized Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins

4 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

5 MG-63 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

6 Coenzyme Q-10 (Ubiquinol) Coenzyme Q-10 Present in most eukaryotic cells Primarily in mitochondria Exists in three redox states Fully oxidized (ubiquinone), partially reduced (semiquinone or ubisemiquinone), and fully reduced (ubiquinol) Suggested anti-cancerous effects Ubiquinol Electron-rich (reduced) form of Coenzyme Q-10 Essential antioxidant in cellular systems Essential component of ATP synthesis Ubiquinol (C 59 H 92 O 4 )

7 Purpose To determine the effects of Coenzyme Q-10 (Ubiquinol) on the proliferation and differentiation of C2C12 stem cells, and the proliferation of MG-63 cancer cells

8 Hypotheses Null Hypotheses Coenzyme Q-10 WILL NOT significantly increase the rate that C2C12 stem cells proliferate and differentiate Coenzyme Q-10 WILL NOT significantly decrease the rate that MG-63 cancer cells proliferate Alternative Hypothesis Coenzyme Q-10 WILL significantly increase the rate that C2C12 stem cells proliferate and differentiate Coenzyme Q-10 WILL significantly decrease the rate that MG-63 cancer cells proliferate

9 Materials Cryotank Three 75mm 2 tissue culture treated flasks Twelve 25 mm 2 tissue culture treated flasks Fetal bovine serum (FBS) C2C12 Myoblastic Stem Cell Line MG-63 Osteosarcoma Cancer Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette tips (1 ml, 5 ml, 10 ml, 20 ml) Micropipettes + sterile tips 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]) Ubiquinol (contents of softgel capsule) 75 ml culture flask Incubator Nikon Inverted Microscope 24 well plate Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Hemacytometer Sterile PBS Ethanol (70% and 100%) Purple Nitrile gloves

10 Procedure: Cell Culturing A 1 ml aliquot of C2C12 cells and MG-63 cells from a Cryotank was used to inoculate 30 ml of 10% serum DMEM media in two 75mm 2 culture flasks yielding a cell density of approximately 10 6 to 2x10 6 cells/ml. The media was replaced with 15 ml of fresh media to remove cryo-freezing fluid and incubated (37 C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/ml was reached. The culture was passed into two sets of 3 flasks in preparation for the experiment and incubated for 2 days at 37 C, 5% CO 2.

11 Procedure: Proliferation Experiment - Day 0 (Addition of Variable) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/ml). 0.1 ml of the cell suspension was added to six 25 mm 2 tissue culture treated flasks containing 6 ml of DMEM (com) media, creating a cell density of approximately 10 5 cells per flask. The stock solution of ubiquinol (1000X) was created using 10 ml of ethanol and the contents of one softgel capsule (containing 200 mg of the active ingredient, Ubiquinol). The following concentrations of variable (next page) were added to the flasks, with 6 flasks for each cell line (two for each concentration of variable). The cells were incubated at 37 C, 5% CO 2 for the remainder of the study.

12 Concentrations of Variable Proliferation Experiment: Differentiation Experiment: 0 (Control) X (Low) 10X (High) 0 (Control) X (Low) 10X (High) Stock 0 ml 6 µl 60 µl Media 6 ml ml ml Stock 0 ml 1 µl 10 µl Media 1 ml ml ml Total 6 ml 6 ml 6 ml Total 1 ml 1 ml 1 ml X equals the estimated concentration of the variable present in the fluid compartments of the body.

13 Procedure: Proliferation Experiment - Days 1 and 4 Day 1 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 Hemacytometer for quantification (eight counts per flask). Day 1 and Day 4 The previous procedure for determining densities was used again, and a Nikon Inverted Microscope was used to take images of representative areas of each flask.

14 Procedure: Differentiation Experiment Day 0 1 ml of C2C12 cells from the 75mm 2 flask was plated into four wells per group, for a total of 12 wells on the 24 well plate. 1 ml of DMEM (com) media was then added to all 12 wells, reaching a total of 2 ml per well. Day 4 The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation. The previously shown concentrations of variable were added to the wells. Day 4, 6, 8, and 10 Using the Nikon Inverted Microscope, images of representative areas of each of the groups were taken.

15 Statistical Analyses of the Proliferation Results ANOVA Compares variation within groups to variation between groups Using the ANOVA, if a p-value less than the alpha of 0.05 is generated (significant variation), it suggests that the null hypothesis can be rejected Dunnett s Test Compares each experimental group to control individually 0.05 alpha was used, and each generated T-value was compared to the T-critical value of 2.67

16 Cell Count (cells per flask) Results of Proliferation Analysis (C2C12) Day 1 - P-value: 6.08x10-13 Day 4 - P-value: 6.94x Control Low High (0) (X) Concentration (10X) Day 1 Day 4

17 Cell Count (cells per flask) Results of Proliferation Analysis (MG-63) Day 1 - P-value: 5.73x10-8 Day 4 - P-value: 5.09x Day 1 Day Control Low High (0) (X) Concentration (10X)

18 Dunnett s Test Results: Day 1 Concentration T-Value T-Critical (α = 0.05) Variation C2C Low (X) Insignificant High (10X) Significant MG Low (X) Insignificant High (10X) Significant

19 Dunnett s Test Results: Day 4 Concentration T-Value T-Critical (α = 0.05) Variation C2C Low (X) Significant High (10X) Significant MG Low (X) Significant High (10X) Significant

20 Differentiation Results: Day 4 Control Low High

21 Differentiation Results: Day 6 Control Low High

22 Differentiation Results: Day 8 Control Low High

23 Differentiation Results: Day 10 Control Low High

24 Conclusions Proliferation C2C12 Based upon the results gathered from the ANOVA and Dunnett s statistical analyses, it appears that the addition of Ubiquinol in low concentration significantly affected stem cell proliferation on Day 4 (positively), and in high concentration significantly affected stem cell proliferation on Days 1 and 4 (negatively). MG63 Based upon the results gathered from the ANOVA and Dunnett s statistical analyses, it appears that the addition of Ubiquinol in low concentration significantly affected cancer cell proliferation on Day 4 (positively), and in high concentration significantly affected cancer cell proliferation on Days 1 and 4 (negatively). Differentiation It appears as if Ubiquinol does affect myotube differentiation (positively in low concentration, but negatively in high concentration)

25 Future Changes Limitations Hemacytometer counts in proliferation subject to technique/clumping errors Differentiation test was qualitative Possible toxic effect of Ubiquinol Extensions Obtain an LD-50 for Ubiquinol Quantitative proliferation (MTT assay, etc.) Use a wider range of concentrations Test using different forms of Coenzyme Q-10 Synergistic effects?

26 Works Cited Bellomo, David. The Stem Cell Divide. New York: AMACOM, Print. Drapeau, Christian. Cracking the Stem Cell Code. Vancouver, Washington: Sutton Hart Press, Print. Fox, Cynthia. Cell of Cells: The Global Race to Capture and Control the Stem Cell. New York: W.W. Norton & Company, Print. National Research Council and Institute of Medicine. Stem Cells and the Future of Regenerative Medicine. Washington, D.C.: National Academy Press, Print Park, Alice. The Stem Cell Hope: How Stem Cell Medicine Can Change Our Lives. New York: Hudson Street Press, Print Mummery, Christine. Stem Cells: Scientific Facts and Fiction. Boston: Academic Press, Print. Scott, Christopher Thomas. Stem Cells Now. New York: Pi Press, Print.