Foundation Louis Stokes Alliances for Minority Participation grant (Award No. 1305001). Figure 2. Reaction to create the acryloyl chloride-cysteine compound ...
Cytotoxicity of Acryloyl Chloride Derivatives on ARPE-19 Cells Katelyn
3 Feltner ,
Kaiya
2 Flemons ,
Westley
4 Cruces ,
George
4 Negrete
and Andrew T.
1 Tsin
1
Department of Biology, The University of Texas at San Antonio, San Antonio, TX, U.S.A. 2 Department of Chemistry, Oakwood University, Huntsville, AL, U.S.A. 3 Department of Biology, Palo Alto College, San Antonio, TX, U.S.A. 4Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX, U.S.A.
Abstract
Methods
Citrus greening disease is caused by a bacterial infection that has proven to be detrimental to citrus trees and plants across Africa, Asia, and most recently the southeastern United States. Citrus greening causes citrus trees to die within several years of being infected and makes their fruit unsuitable for sale.2 Our collaborator has synthesized acryloyl chloride derivatives that can be used to treat bacterial diseases that directly affect the agricultural industry, such as citrus greening. We are treating adult retinal pigmented epithelial (ARPE-19) cells to determine if these derivatives are toxic to human cells as well as bacteria. This experiment is a structure-activity relationship (SAR) study between the structure of acryolyl chloride-derived analogs (ACAs) and their bioactivity in ARPE-19 cells. These compounds share a thiazolidine core with an acryloyl group that can be coupled with different functional groups such as: alkyl thiols, aryl thiols, carboxylic acids, or aryl nitro groups. These different functional groups may change the biological behavior of the particular ACA. In order to test the bioactivity of each ACA, we measured the cell proliferation of ARPE-19 cells, treated with different concentrations of each ACA. We hypothesized that treatment of ARPE-19 cells with acryolyl chloride-derived analogs would result in cell toxicity causing a decrease in cell proliferation.
Conclusion
The acryloyl chloride-derived analogs (ACAs) were made by combining methanol, 3-nitrobenzaldehyde, potassium carbonate, cysteine, and a specific acryloyl chloride derivative. In order to test the bioactivity of these acryloyl chloride-derived analogs (ACAs) on cells, first a growth curve had to be determined. ARPE-19 cells were obtained from the American Type Culture Center. The ARPE-19 cells were seeded at 20,000 per well and incubated for 24 hours. Cells were harvested at 0, 24, and 48 hours. A cell count was performed at each time mark using the Neubauer Hemacytometer and trypan-blue exclusion method1. ARPE-19 cells were seeded again at 20,000 per well and incubated for 48 hours. MC was dissolved in water and sodium bicarbonate, then introduced to the cell media using Hank’s Balanced Salt Solution (HBSS) 1x. MC + EtOH + SH, MC + MEOC(=O)Cl, and MC + Oc + SH were all dissolved in dimethyl sulfoxide (DMSO) and introduced to the cells at 0.01% DMSO in serum free DME/F12 media. One set of cells were treated with 0nM, 500nM and 1μM of MC or MC + EtOH + SH. Another set of cells were treated with 0nM, 100nM and 1μM of MC + Oc +SH or MC + MeOC(=O)Cl. Cells were harvested at 0, 24, and 48 hours. A cell count was performed at each time mark using the Neubauer Hemacytometer and trypan-blue exclusion method.
Figure 2. Reaction to create the acryloyl chloride-cysteine compound (MC) from which three other novel analogs were derived.
Results
Our results oppose our original hypothesis that cell growth would drastically decrease in all of the novel compounds tested. At concentrations of 100nM and 1μM both MC+Oc+SH and MC+MeOC(=O)Cl had consistent trends with the normal cell growth of ARPE-19 cells, excluding the 100nM treatment with MC+Oc+SH, which can be attributed to mechanical error, thus several more trials are needed. Both compounds showed a slower increase in viable cell number than untreated cells possibly suggesting a higher concentration of these compounds is needed to see toxic behaviors on our ARPE-19 cells. Because the 0nM treatments of MC and MC + EtOH + SH did not follow the trend of normal cell growth, the data gathered is inconclusive and further testing must be done. Further investigation of these compounds is needed at higher concentrations on animal and bacterial cells in order to better understand any adverse effects these derivatives may produce on cell growth.
References
Introduction In our effort to better understand the bioactivity of acryloyl chloride derivatives, we have worked in collaboration with the organic chemistry department to synthesize three novel analogs. The altered chemical structures of these derivatives are each unique and their effects on cell activity are unprecedented. This study may be helpful in determining the cytotoxicity of these analogs on ARPE-19 cells, leading to further research on the possible pharmaceutical or agricultural uses of these compounds. .
A
1Betts
BS, Parvathaneni K, Yendluri BB, Grigsby J, Tsin AT. Ginsenoside-Rb1 Induces ARPE-19 Proliferation and Reduces VEGF Release. ISRN Ophthalmol. 2012 Jan 4;2011:184295. PMID: 24527228 2Massenti, Figure 3. Viable Cell Number after 48h treatment with various concentrations of MC. At 0h, all concentrations had 16,667 viable cells. At 24h, 0nM increased to 23,704 cells, 500nM increased to 20,000 cells and 1μM increased to 22,593 cells. At 48h, 0nM decreased to 20,000 cells, 500nM increased to 32,963 cells, and 1μM to 22,222 cells.
Figure 4. Normal Cell Growth. Cells were seeded at 20,000 cell per well and then counted at 0h, 24h, and 48h. At 0h, there were 9,259 cells. At 24h, there were 20,000 cells, and at 48h there were 45,923 cells.
Figure 5. Viable Cell Cumber after 48h treatment with various concentrations of MC+MeOC(=O)Cl. At 0h, all concentrations had 16,667 viable cells. At 24h, 0nM increased to 17,777 cells, 100nM increased to 18,888 cells, 1μM decreased to 16,295 cells, and the vehicle control (0.01% DMSO) decreased to 16,666 cells. At 48h, 0nM increased to 26,296 cells, 100nM increased to 19,258 cells, 1μM increased to 23,333 cells, and the vehicle control increased to 21,851 cells.
Acknowledgments
C
B Figure 6. Viable Cell Number after 48h treatment with various concentrations of MC + SHEtOH. At 0h, all concentrations had 16,667 viable cells. At 24h, 0nM increased to 23,704 cells, 500nM increased to 20,000 cells, 1μM increased to 22,593 cells, and the vehicle control (0.01% DMSO) increased to 18,889 cells. At 48h, 0nM decreased to 20,000 cells, 500nM increased to 32,963 cells. At 1μM to 22,222 cells and the vehicle increased to 18,889 cells.
Figure 1. Acryloyl chloride derived analogs.
R., Lo Bianco, R., Sandhu, A. K., Gu, L., & Sims, C. Huanglongbing modifies quality components and flavonoid content of 'Valencia' oranges. J Sci Food Agric. 2014 Dec 29 PMID: 25546309
Figure 7. Viable cell number after 48h treatment with MC+Oc+SH . At 0h, all concentrations had 16,666 viable cells At 24h, 0M had 17,036 cells, 100nM had 19,629 cells, 1μM had 16,296 cells, and the vehicle control (0.01% DMSO) had 16,666 cells. At the 48h, 0M had 26,296 cells, 100nM had 19,258 cells, 1μM had 23,333 cells, and the vehicle control had 21,851 cells.
Authors thank UTSA Summer Research Program, CIMA-LSAMP program, and the IMARI grant from Oakwood University. This project was supported by a grant from the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health. Funding also provided by a National Science Foundation Louis Stokes Alliances for Minority Participation grant (Award No. 1305001)
