by James Babo (Queensborough CC, Biology, 2022-2023 CRSP cohort)
The work was done as a part of the CRSP program at Queensborough Community College/CUNY, under the supervision of Dr. Regina Sullivan.
This article has been published as part of the Special Edition of Ad Astra, which features the CUNY Research Scholars Program (CRSP) across The City University of New York. The issue is accessible at http://adastraletter.com/2024/crsp-special-edition/.
ABOUT THE AUTHOR
James Babo
James Babo is a Clinical Laboratory Science major at Hunter College. In 2023, he obtained an associate degree in Biological Science from Queensborough Community College, where he participated in the CRISP program. While at Queensborough, he also took part in the "Becoming a Scientist" program at Rockefeller University, designed for CUNY community college students, and learned about research ethics and advanced lab techniques. Currently, he is working as a research apprentice at a neuroscience lab at Albert Einstein College of Medicine. He enjoys riding bicycles, learning new languages, trying new restaurants, and producing music in his free time. James is always eager to learn new things and challenge himself.
Lung cancer is the primary cause of cancer-related deaths in the US. Single-walled Carbon Nanotubes (SWCNT) are stable nanotube size tubes of carbon with the potential for delivering drugs to cancerous cells. Our research aims to understand the effect of unfunctionalized pristine Single-walled Carbon Nanotubes (SWCNTs) on the rate of migration and the viability of A549, a lung cancer cell line. Endothelial cells were used as non-cancer cell control in viability studies. Our preliminary results indicate that SWCNTs reduces the migration rate of non-small lung cancer cells, which is crucial in preventing metastasis. The MTT assay results show that at a concentration of 12.5 μg/ml SWCNT showed about 50% cytotoxicity. However, Trypan Blue viability studies show little to no cytotoxicity in similar SWCNT concentration ranges indicating there may be a specific mitochondrial effect, since the MTT assay relies on the functionality of a mitochondrial enzyme. SWCNT treatment did not reduce endothelial cell viability suggesting an effect specific to lung cancer cells.
Carbon nanotubes (CNTs) were discovered by Sumio Iijima in 1991 (Iijima, 1991). They have a unique hexagonal carbon atom arrangement and exhibit remarkable properties. There are two types of CNTs: single-walled (SWCNTs) and multi-walled (MWCNTs). These nanotubes have various applications in electronics, materials science, medicine, and defense (Prato. Et al, 2008).
In a study of the effect of SWCNT on cytoskeletal dynamics, mainly focusing on actin structures, researchers showed that SWCNT influences actin filaments (Holt et al., 2010). Actin filaments are essential cytoskeleton components, providing structural support and facilitating cellular movement. Through fluorescence microscopy and actin polymerization assays, the authors demonstrate that SWCNTs can modulate actin nucleation and elongation rates, ultimately reorganizing actin structures within cells.
Based on these findings, our hypothesis is that carbon nanotubes will influence the migration rate of lung cancer cells.
Our study used A549, a non-small cell lung cancer cell line and endothelial cells as a non-cancer cell control.
A549 cells, a non-small cell lung cancer cell line, were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin and fungizone at 37°C and 5% CO2. Endothelial cells were a gift of Dr. Rochelle Nelson, (QCC Biology Dept). These cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin and fungizone at 37°C and 5% CO2.
A solution containing 1 mg/ml of SWCNTs in complete Dulbecco's Modified Eagle Medium (DMEM) was prepared and sterilized under UV light for 30 minutes. The solution was sonicated on ice for an hour to disperse the nanotubes evenly and then vortexed five times immediately before cell treatment to ensure uniform distribution.
For the scratch assays also called wound healing assays, A549 cells were grown to a confluent monolayer. Then the cells treated with 0, 6.25, 12.5, 25, 50 μg/ml for 24 hours, the medium was removed and replaced with DMEM containing 5% serum to reduce cell division. The reduced serum DMEM contained the same concentrations of SWCNT as above. A wound was introduced using a 200 μL pipette, and imaging was done at 0, 24 hours, and 48 hours to monitor the effects of the SWCNT treatment on cell migration.
The Trypan Blue assay was conducted to assess cell death. In this assay, cells were treated with SWCNT in concentrations of 0,6.25, 12.5,25,50 μg/ml for 24 hours solution. Cells were trypsinized then stained with Trypan Blue dye, which is taken up by dead cells. The number of stained cells was then counted a phase contrast microscope and a hemocytometer to determine the percentage of viable cells.
The MTT assay was used to evaluate cell viability. A549 cells or Endothelial cells were treated with SWCNT in concentrations of 0,12.5,25,50,100,200 μg/ml for 24 hours and then incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 hours. MTT is converted into a colored formazan product by metabolically active cells. The plate was read with a standard plate reader at 570 nm.
Migration Assay. The results from the wound healing assay indicate a notable effect of SWCNT (single-walled carbon nanotubes) exposure on cellular behavior. Specifically, the assay revealed reduced migration rates following exposure to 50 μg/ml of SWCNT. After 24 hours, there was a 10% decrease in compared to the control group, suggesting an inhibitory effect on cellular proliferation. Furthermore, after 48 hours, the reduction in cell migration was even more pronounced, with a 20% decrease observed compared to the control group.
Figure 1: Migration assay taken at 0hr, 24hr, 48hr.
MTT Assay. In our laboratory's previous results, the effects of different concentrations of single-walled carbon nanotubes (SWCNT) on cell viability was investigated. The results showed that even a low concentration of 12.5 μg/ml of SWCNT can lead to a significant reduction of 50% in cell viability.
Figure 2: MTT assay taken at concentrations 0-200 μg/ml.
Trypan blue assay. The results of the trypan blue assay showed there was no observable difference in the rate of cell proliferation when exposed to SWCNTs. in of concentrations of 0, 6.25, 12.5, 25, 50 μg/ml.
Figure 3: Trypan blue assay at concentrations 0-50 μg/ml.
MTT assay with Endothelial cells. After SWCNT treatment in concentrations of 0, 5, 10, 20 and 40 μg/ml, of endothelial cells showed no reduction in cytotoxicity.
Figure 4: MTT assay at concentrations of 0-40 μg/ml..
SWCNTs' effect on lung cancer cells revealed that exposure to low SWCNTs (12.5 μg/ml) caused a significant 50% reduction in cell viability, as shown by the MTT assay. This finding highlights the potential of SWCNTs in inhibiting tumor growth by negatively impacting cell survival. However, the Trypan Blue assay produced contradictory results, indicating no significant difference compared to the control in cell viability after SWCNTs treatment. This may be due to the different mechanism of the assays. To show cellular viability the MTT assay requires the activity of the NADH-dependent cellular oxidoreductase enzymes while the trypan blue assay requires the integrity of the plasma membrane. These results point to a possible clue to as to how SWCNT treatment affects A549 cells. These preliminary suggest SWCNTs may affect the mitochondrial metabolic activity of A549 cells.
After SWCNT treatment endothelial cells showed little to no cytotoxicity. The result supports a possible differential effect on the A549 cancer cells and a normal cell line.
Although much more experimentation is required our results show that SWCNT may have potential as a unique anti-cancer agent.
Prato, M., Kostarelos, K., Bianco, A. (2008). Functionalized carbon nanotubes in drug design and discovery. Accounts of Chemical Research. Available online: https://pubs.acs.org/doi/abs/10.1021/ar700089b.
“Seer Cancer Stat Facts.” SEER, 2017. Available online: http://seer.cancer.gov/statfacts.
Iijima, S. (1991). Helical microtubules of graphitic carbon.
Mao, H., Kawazoe, N., Chen, G. (2013). Uptake and intracellular distribution of collagen-functionalized single-walled carbon nanotubes. Biomaterials, 34(10), 2472-2479.
Holt, B. D., Short, P. A., Rape, A. D., Wang, Y. L., Islam, M. F., Dahl, K. N. (2010). Carbon nanotubes reorganize actin structures in cells and ex vivo. ACS Nano, 4(8), 4872-4878. Available online: https://pubmed.ncbi.nlm.nih.gov/20669976/.