Narcan Effects on Cancer Cell Behavior Conor Maddigan

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 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 • NARCAN Nasal Spray • Two 75 mm 2 tissue culture treated flasks • 75 ml culture flask • Twelve 25 mm 2 tissue culture treated flasks • Incubator • Fetal bovine serum (FBS) • Nikon Inverted Microscope • MG 63 Osteosarcoma Cancer Cell Line • Laminar Flow Hood with UV Sterilizing Lamp • Macropipette + sterile macropipette tips (1 m. L, 5 m. L, 10, m. L, 20 m. L) • Labeling Tape • Micropipettes + sterile tips • Sterile PBS • DMEM Serum - 1% and Complete Media (4 m. M L-glutamine, 4500 mg/L glucose, 1 m. M sodium pyruvate, and 1500 mg/L sodium bicarbonate + [10% fetal bovine serum for complete]) • Hemocytometer • Ethanol (70%) • Purple Nitrile gloves • Trypsin-EDTA • Pen/paper

Procedure 1. A 1 m. L aliquot of MG 63 cells from a cryotank were used to inoculate 30 m. L of 10% serum DMEM media in a 75 cm 2 culture flask. 2. The media was replaced with 15 m. L of fresh media to remove the cryo-freezing fluid and incubated (37 C, 5% CO 2) for 2 days. 3. The culture was passed into 2 flasks in preparation for the experiment and incubated for 2 days at 37 C, 5% CO 2.

Procedure 1. After trypsinization, cells from both of the flasks were pooled into 1 common 75 cm 2 flask. 2. 1 ml of the cell suspension, 4 ml of media, and the appropriate variable were added to 12 25 cm 2 tissue culture treated flasks. 3. The following concentrations (next page) were added to each of the flasks. 4. 2 flasks per group, per day (2 days). 5. The cells were incubated (37 C, 5% CO 2) for the remainder of the study.

Experimental Groups Concentrations Control Low (0. 000004%) High (0. 0004%) Cell suspension 1 ml (10 x 105 per ml) 1 ml The high concentration (x) was created by diluting the solution. Media 4 ml The low concentration was created through a 1: 100 dilution of the high concentration. Narcan Variable 0 ul 30 ul Standard dose: 4 mg A stock solution was created by mixing 2 mg of Narcan in 50 ml of media, making a 100 x stock solution.

Counting Procedure 1. The Day 1 flasks were trypsinized. 2. 2 mls of media were added. 3. The cells were then counted by aliquoting 20 ul of the cells from the counting flask onto each grid on a hemocytometer. 4. This was done eight times per flask, resulting in sixteen counts for each concentration per day.

Cell counts (*105) Narcan Effects on MG 63 Cells: Day 1 50 45 40 35 30 25 20 15 10 5 0 Day 1 p-value: 0. 000024 Control Low (0. 000004%) Narcan concentrations High (0. 0004%)

Dunnett’s Test: Day 1 Low High T-crit T-value Result 3. 22 0. 36 Not Significant 3. 22 4. 33 Significant

Cell Pictures: Day 1 Control Low High

Narcan Effects on MG 63: Day 2 120 Cell counts (*105) 100 Day 2 p-value: <0. 0001 80 60 40 20 0 Control Low (0. 000004%) Narcan concentrations High (0. 0004%)

Dunnett’s Test: Day 2 T-crit T-value Result Low 3. 22 1. 09 Not Significant High 3. 22 6. 25 Significant

Cell Pictures: Day 2 Control Low High

Conclusion • A high concentration of Narcan caused a significant effect on MG 63 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 • It is unlikely that all cell suspensions were completely homogeneous. • Trypsinizing the flasks and some pipetting were not fully synchronized. • Potential errors in counting. Possible Extensions • Test Narcan on different cell lines (C 2 C 12, 3 T 3). • Different exposures of Narcan on MG 63 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. MB 231 cells. ” January 3 2018. https: //www. ncbi. nlm. nih. gov/pmc/articles/PMC 5757202/#__ffn_sectitle. La. Morte, Wayne W. “Characteristics of Cancer Cells. ” Prospective versus Retrospective Studies, 4 May 2016, sphweb. bumc. bu. edu/otlt/MPH-Modules/PH/PH 709_Cancer 7. 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_af 4824 • 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