Pancreatic cancer is usually a fatal type of cancer and the prognosis for patients is poor, as there is a 91% probability of dying within 5 years following diagnosis. Thus, early detection methods of screening are crucial for the treatment of pancreatic cancer. Previously, two pancreatic cancer (Mia Paca-2) targeting peptides MCA1 and MCA2 were discovered via phage display technology. These peptides were found to bind specifically to pancreatic cancer cells. The objective of this study was to determine the binding affinity by measuring the half-maximal effective concentration (EC50) of MCA1 and MCA2, the specific binding by measuring the half-maximal inhibitory concentration (IC50), and to evaluate their effects on pancreatic cancer cell viability. I utilized a modified enzyme-linked immunosorbent assay (ELISA) to determine the EC50 of MCA1 and MCA2 against Mia Paca-2, ovarian cancer (SKOV-3), prostate adenocarcinoma (LNCaP), human embryonic kidney (HEK 293), and human normal pancreatic (hTERT-HPNE) cells. I added various concentrations of the peptides (0.1–300 μM) to the cells and then incubated them for 1 hr at 37° C and 5% CO2. I then washed unbound peptide with 1% bovine serum albumin (BSA), fixed cells with 10% formalin, blocked with 10% FBS, 0.3 M glycine, and 0.05% Tween-20 in PBS, and incubated with horseradish peroxidase-conjugated streptavidin (HRP). I measured the absorbances at 405 nm after adding azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) substrate. MCA1 and MCA2 binding followed a sigmoidal dose-response curve against Mia-Paca-2 cells, from which it was possible to determine the EC50 values, which were 16.11 ± 8.91 µM and 97.01 ± 4.88 µM (mean ± SEM), respectively. A comparison between the binding ratio of these peptides with Mia Paca-2, LNCaP, HEK 293 cells for MCA1, and HEK293 cells for MCA2 showed minimal binding. However, no binding was found for SKOV-3 and hTERT-HPNE cells for MCA1 or LNCaP, SKOV-3, and hTERT-HPNE cells for MCA2. Additionally, the dose-response curves were not well-fitted for SKOV-3, HEK293, LNCaP, and hTERT-HPNE cell lines. I conducted a competitive modified ELISA to determine the IC50 of biotin-GSG-MCA1 and biotin-GSG-MCA2 peptide to Mia Paca-2. I added varying concentrations of GSG-MCA1 or GSG-MCA2 (0.03–100 µM) to cells propagated to 80% confluency on a 96-well plate and preincubated for 15 min at 37° C. I then added biotin-GSG-MCA1 or biotin-GSG-MCA2 peptide (30 μM) into the preincubated wells for 1 hr. I conducted the modified ELISA as previously described. MCA1 and MCA2 competitive binding followed a sigmoidal dose-response curve against Mia-Paca-2 cells, from which it was possible to determine the IC50 values, which were 2.15 ± 1.38 µM and 36.09 ± 1.54 µM (mean ± SEM), respectively. I evaluated the effect of these peptides on cell viability of pancreatic cancer cells utilizing an (3-(4,5 dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) assay. I incubated Mia Paca-2 cells with 10μM peptides or DMSO, then added 10µL of MTT reagent. After a 4 hr incubation period, I measured their absorbances spectrophotometrically at a wavelength of 570 nm. No observable decrease in cell viability was seen for the Mia Paca-2 cells treated with MCA1 or MCA2 when compared to the DMSO control. Peptides MCA1 and MCA2 exhibit binding affinity and specificity to pancreatic cancer cells with EC50 and IC50 values in the μmolar range, but do not have any effect on cell viability of Mia Paca-2 cells. Therefore, these peptides may be used for early detection of pancreatic cancer to improve the overall outcome of the disease.
|Commitee:||Hum-Musser, Sue, Holt, Scott|
|School:||Western Illinois University|
|School Location:||United States -- Illinois|
|Source:||MAI 82/1(E), Masters Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Dose response, Molecular targeting, Pancreatic cancer, Peptide, Phage display|
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