The Effect of Epinephrine on Cancer Cell Proliferation. Alec Chrystal Grade 11 Central Catholic High School PJAS 2019

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1 The Effect of Epinephrine on Cancer Cell Proliferation Alec Chrystal Grade 11 Central Catholic High School PJAS 2019

2 Stress is the feeling of physical, social, or emotional pressure Struggling to meet deadlines Psychological Stress There are two main causes of stress Routine Events Unusual Events A car cutting you off on the highway Death or illness of immediate family member Trauma

3 A Natural Response The human body is wired to react to stress in a way that protects it from aggressors. Upon encountering the stressor: 1. Nerve signals stimulate the adrenal glands 2. Adrenal glands release various hormones, including epinephrine 3. Results in elevated heart rate, blood pressure, and energy supplies. This stress response is still activated today.

4 Chronic Stress Typically, after a threat has passed hormone levels return to normal. When stressors are constantly present, this stress response stays turned on. Chronic Stress has been linked to increased risk of: ØAnxiety ØDepression ØHeart Disease ØMemory and Concentration Impairment ØPossibly Cancer According to the American Psychological Association 44% of Americans Suffer from Chronic Stress Monitor on Psychology, January 2011, Vol. 42, No. 1

5 Epinephrine Epinephrine is a hormone and neurotransmitter produced by the adrenal glands. Causes an adrenergic receptive mechanism on effector cells Mimics actions of the sympathetic nervous system.

6 Stress and Cancer Multiple studies have established a link between stress and cancer. In one study, tumors were transplanted into the mammary fat pads of mice. There was a significantly higher rate of metastasis in mice who were chronically stressed compared to those who were not stressed.

7 Research Objective The objective of this research was to determine if stress has an effect on cancer cells.

8 MG-63 Cancer Cells Cancer cells are cells that grow and divide at an unregulated rate. Apoptosis does not occur in cancerous cells. MG-63 Cells are derived from human Osteosarcoma cells, an aggressive form of bone cancer. MG-63 cells are a useful cell model for proliferation focused cancer research.

9 Purpose of Experiments To determine how epinephrine affects the proliferation of MG-63 cells.

10 Hypotheses 01 Null Hypothesis: Epinephrine will not have a significant effect on the proliferation of MG-63 cancer cells. 02 Alternative Hypothesis: Epinephrine will have a significant effect on the proliferation of MG-63 cancer cells.

11 Materials 25cm 2 culturing flasks 75cm 2 culturing flasks Trypsin Hemocytometers 10% DMEM media- (4mLglutamine, 45000mg/L gluconse, 1mL sodium pyruvate, and 1500mg/L sodium bicarbonate + 10% fetal bovine serum) PBS P1000 pipette P100 pipette Sterile Tips Micro pipette Macro pipettes Incubator EVOS Computer Imaging System Laminar Sterilized Hood Sterile Gloves 70% Ethanol MG-63 Cells Pen and notebook Epinephrine (Variable)

12 Procedure- Cell Culture A 1 ml aliquot of MG63 cells from a cryotank was used to inoculate 30 ml of 10% DMEM media into two 75cm 2 culture flasks. The media was replaced with 15 ml of fresh media to remove the cryo-freezing fluid and incubated (37 C, 5% CO 2 ) for 2 days. The culture was passed into 2 sets of 3 T75 flasks in preparation for the experiment and incubated for 2 days.

13 Procedure Day 0 After trypsinization, cells from all the flasks were pooled into one common 75cm 2 flask 1 ml of cell suspension was added to twelve T25 experimental flasks containing 4mL of 10% DMEM media. The following concentrations of variable (next page) were added to their respective flasks. The cells were incubated at 37C, 5% CO 2 for the remainder of the study.

14 Concentrations Standard Dose: 0.01g/kg A stock solution was created by mixing 40mg of epinephrine in 50mL of ethanol, creating a 1000x stock concentration. A high concentration (X) was created through a 1:100 serial dilution of the stock solution. A low concentration (0.01X) was created through a 1:100 dilution of the high concentration. Control Low High Concentration 0% % % Cell Suspension 1mL 1mL 1mL Media 4mL 4mL 4mL Variable (Epinephrine) 0uL 30uL 30uL

15 Procedure- Counting (Days 1 and 2) The media in the T25 flasks was removed 1mL of trypsin was added to the T25 flasks and then immediately removed 500uL of fresh trypsin was added into the flasks. The flasks were then incubated for 4 minutes The flasks were then slapped to create a cell suspension and was confirmed by microscopy. 20uL aliquots were transferred to a hemocytometer from quantification (eight counts per flask)

16 Proliferation Images Day 1 Control Low High

17 Proliferation Images Day 2 Control Low High

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19 Dunnett s Test- Day 2 Variable (compared to control) T Value (T-Crit=3.22) Relevance Low 3.40 Significant High 2.32 Not Significant Alpha: 0.01

20 The null hypothesis in respect to day 2 counts can be rejected, indicating that epinephrine significantly increased proliferation of MG-63 cancer cells over time. Hypothesis Result and Potential Explanation Theoretically, epinephrine should have induced a proliferative effect on the MG-63 cancer cells, as alpha 1 adrenergic receptors exist in this cell line. [Biochemical and Biophysical Research Communications, Nov. 2012] [Journal of Cellular Physiology, Jul. 2009] However, this was only demonstrated in the Day 2 Low counts in this study. Past research has indicated that alpha-1 agonists induced downregulation of aortic smooth muscle alpha 1-receptors by 70% after 26 hours. [Life Sciences, Oct. 1983] It is plausible that epinephrine could have induced a similar effect in the Day 2 high count of MG-63 cells, resulting in non-significance.

21 Experimental Conclusion The objective of this experiment was to determine if epinephrine had a proliferative effect on MG-63 cancer cells. The data suggests that epinephrine caused a significant increase in cancer cell proliferation over a period of time.

22 Research Conclusion Chronic stress has a proliferative effect on cancer cells over time.

23 Limitations Cell counting was subject to human error. Epinephrine was not available in its purest form. More replicates could have been used. Short observation period. Only cell counts were performed to test the effect of epinephrine.

24 Further Studies Studies to determine why Day 1 counts were not significant A more specific range of concentrations could be used (0.01X, 0.001X) Test the effects of epinephrine on various cell lines such as C2C12 Stem Cells. A combination of multiple stress hormones such as cortisol, epinephrine, and norepinephrine. Other cell parameters could be studied to determine the effect of epinephrine on cancer cells.

25 Sources 1. Huang HH, Brennan TC, Muir MM, Mason RS. Functional alpha1- and beta2-adrenergic receptors in human osteoblasts. J Cell Physiol Jul. 2. Luo Q, Wang X, Liu R, Qiao H, Wang P, Jiang C, Zhang Q, Cao Y, Yu H, Qu L. alpha1a-adrenoceptor is involved in norepinephrine-induced proliferation of pulmonary artery smooth muscle cells via CaMKII signaling. J Cell Biochem Dec Lutgendorf SK, DeGeest K, Dahmoush L, et al. Social isolation is associated with elevated tumor norepinephrine in ovarian carcinoma patients. Brain Behav Immun McEwen BS. Stressed or stressed out: what is the difference?. J Psychiatry Neurosci Monitor on Psychology, January Psyc Dataset, vol. 42, no. 1, 2011, p Moreno-Smith M, Lutgendorf SK, Sood AK. Impact of stress on cancer metastasis. Future Oncol Pu J, Bai D, Yang X, Lu X, Xu L, Lu J. Adrenaline promotes cell proliferation and increases chemoresistance in colon cancer HT29 cells through induction of mir-155. Biochem Biophys Res Commun Nov Pyschological Stress and Cancer. National Cancer Institute, National Cancer Institute, 10 Dec Santos IN, Spadari-Bratfisch RC. Stress and cardiac beta adrenoceptors. Stress Jun. Mark Krotec P.T.E.I. Phil Campbell Ph.D.

26 Day 1 and 2 Anovas