Narcan Effects on Cancer Cell Behavior Conor Maddigan Central Catholic High School Grade 11

Opioid Overdose Statistics An estimated 25% of people that are prescribed opioids for pain misuse them. An estimated 5% of people that are prescribed opioids will transition to heroin. Every day in the US, over 130 people die from an opioid overdose.

Narcan Naloxone hydrochloride NARCAN nasal spray is to be used in an emergency setting to reverse an opioid overdose. Does not substitute for emergency medical care. Can be administered every 2-3 minutes. Available without a prescription.

Cancer Cells The growth of these cells happens at a very irregular pace. Apoptosis is very rare in cancer cells. Mutations from cells are passed on from generation to generation, which causes them to cluster and form tumors. Limitless replication potential. Capable of tissue invasion and metastasis.

MG-63 Cancer Cell Line Human cancer cell line Osteosarcoma cells Useful model for testing the effect of variable on cancer cell proliferation.

Research Objective/Purpose The objective of this experiment is to determine if the recommended dosage of Narcan can alter the behavior of cancer cells.

Hypotheses Null: Narcan will not significantly affect cancer cell proliferation and survivorship. Alternative: Narcan will significantly affect cancer cell proliferation and survivorship.

Materials Cryotank Two 75mm2 tissue culture treated flasks Twelve 25 mm2 tissue culture treated flasks Fetal bovine serum (FBS) MG63 Osteosarcoma Cancer Cell Line 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]) NARCAN Nasal Spray 75 ml culture flask Incubator Nikon Inverted Microscope Laminar Flow Hood with UV Sterilizing Lamp Labeling Tape Hemocytometer Sterile PBS Ethanol (70%) Purple Nitrile gloves Trypsin-EDTA Pen/paper

Procedure A 1mL aliquot of MG63 cells from a cryotank were used to inoculate 30mL of 10% serum DMEM media in a 75cm2 culture flask. The media was replaced with 15mL of fresh media to remove the cryo-freezing fluid and incubated (37 C, 5% CO2) for 2 days. The culture was passed into 2 flasks in preparation for the experiment and incubated for 2 days at 37 C, 5% CO2.

Procedure After trypsinization, cells from both of the flasks were pooled into 1 common 75cm2 flask. 1ml of the cell suspension, 4 ml of media, and the appropriate variable were added to 12 25cm2 tissue culture treated flasks. The following concentrations (next page) were added to each of the flasks. 2 flasks per group, per day (2 days). The cells were incubated (37 C, 5% CO2) for the remainder of the study.

Experimental Groups Concentrations Standard dose: 4mg A stock solution was created by mixing 2mg of Narcan in 50ml of media, making a 100x stock solution. The high concentration (x) was created by diluting the solution. The low concentration was created through a 1:100 dilution of the high concentration. Control Low (0.000004%) High (0.0004%) Cell suspension (10x105 per ml) 1 ml Media 4 ml Narcan Variable 0 ul 30 ul

Counting Procedure The Day 1 flasks were trypsinized. 2mls of media were added. The cells were then counted by aliquoting 20ul of the cells from the counting flask onto each grid on a hemocytometer. This was done eight times times per flask, resulting in sixteen counts for each concentration per day.

Narcan Effects on MG63 Cells: Day 1 Day 1 p-value: 0.000024

Dunnett’s Test: Day 1 T-crit T-value Result Low 3.22 0.36 Not Significant High 4.33 Significant

Cell Pictures: Day 1 Low High Control

Narcan Effects on MG63: Day 2 Day 2 p-value: <0.0001

Dunnett’s Test: Day 2 T-crit T-value Result Low 3.22 1.09 Not Significant High 6.25 Significant

Cell Pictures: Day 2 Low High Control

Conclusion A high concentration of Narcan caused a significant effect on MG63 cells. The null hypothesis was rejected for the high concentrations. The null hypothesis could not be rejected for the low concentrations. A possible explanation for these results relates to how Narcan acts upon opioids.

Analysis Limitations Possible Extensions It is unlikely that all cell suspensions were completely homogeneous. Trypsinizing the flasks and some pipetting were not fully synchronized. Potential errors in counting. Test Narcan on different cell lines (C2C12, 3T3). Different exposures of Narcan on MG63 cancer cells. Analyze cell behavior in a different way. Use a different way to count the cells.

References Barbieri, Antonio, Sabrina Bimonte, and Marco Cascella. “The effects of naloxone on human breast cancer progression: in vitro and in vivo studies on MDA.MB231 cells.” January 3 2018. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757202/#__ffn_sectitle. LaMorte, Wayne W. “Characteristics of Cancer Cells.” Prospective versus Retrospective Studies, 4 May 2016, sphweb.bumc.bu.edu/otlt/MPH-Modules/PH/PH709_Cancer/PH709_Cancer7.html. National Institute on Drug Abuse. “Opioid Overdose Reversal with Naloxone (Narcan, Evzio).” NIDA, 4 Apr. 2018, www.drugabuse.gov/related-topics/opioid-overdose-reversal-naloxone-narcan-evzio. “What Is NARCAN® (Naloxone) Nasal Spray.” NARCAN® (Nalaxone HCl) Nasal Spray, www.narcan.com/.

Pictures https://www.rndsystems.com/products/human-lrp-1-cluster-iii-antibody_af4824 https://www.mbaskool.com/business-concepts/statistics/7430-dunnetts-test.html https://news.psu.edu/story/493940/2017/11/13/research/penn-state-projects-take-opioid-epidemic https://www.schoolhealth.com/narcan-opiate-antagonist-nalaxone-hci-4-mg-spray-bottle-2-pack

ANOVA: Day 1

ANOVA: Day 2