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Determination of Transcriptional Changes Induced by an Ovarian Cancer Targeting Peptide
Author(s) -
Weintraub Lindsey,
Rudy James,
HumMusser Susan,
Musser Richard,
Soendergaard Mette
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.530.16
Subject(s) - ovarian cancer , viability assay , peptide , cytotoxic t cell , apoptosis , cancer research , cancer , chemistry , microbiology and biotechnology , biology , medicine , biochemistry , in vitro
The fifth leading cause of cancer‐related deaths among women is ovarian cancer. Current standard treatment involve cytoreductive surgery, as well as platinum and taxane based chemotherapy. While most patients initially respond to therapy, the majority relapses with drug resistant malignancy, and ultimately succumbs to the disease. Bacteriophage (phage) display technology was previously used to discover a 15‐mer peptide (J18) with specific binding to human ovarian adenocarcinoma (SKOV‐3) cells. Based on the ovarian cancer binding properties of J18, the cytotoxic properties of the peptide was investigated using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. In short, 0.25 mg/mL peptide J18, peptide p30‐1 (non‐cytotoxic), or DMSO were incubated with 10 3 SKOV‐3 cells for 48 h, after which MTT reagent was added. The cell viability was quantified spectrophotometrically at 560 nm. These results showed that J18 significantly (p<0.01) decreased SKOV‐3 cell viability compared to peptide p30‐1 and DMSO. Next, the mechanism behind the loss of cell viability was sought elucidated by investigation of transcriptional changes after addition of peptide J18. Specifically, genes involved in cancer progression ( myc , elk‐1 , smad4 , hspb1 , and tp53 ), and apoptosis ( fadd , casp3 , and cflar ) as well as housekeeping ( sdha ) was chosen for analysis. In brief, SKOV‐3 cells (10 5 ) were incubated with 0.25 mg/mL J18 for 48 h, after which total RNA was extracted using the TRIzol method. Total RNA was used for cDNA synthesis with oligodT primers. Gene specific primers for selected genes were designed using Primer3 software. qPCR reactions were carried out using a SYBR Green Master Mix in optical 96‐well plates on an Applied Biosystems StepOnePlus Real‐Time qPCR System. Sdha was used as the endogenous control to correct for sample variation. The comparative quantification method deltadelta Ct (ΔΔCt) was used, and data were transformed to absolute values with 2‐ΔΔCt to obtain fold changes between treatments. Each treatment consisted of five biological replicates. Data were analyzed with ANOVA then Fisher's LSD at p<0.05. Results showed that expression of myc , elk1 , smad4 , hspb1 , tp53 , fadd , casp3 , and cflar were not significantly different for peptide J18 compared to DMSO. These results indicate that the loss of cell viability mediated by peptide J18 is not associated with changes to the transcriptional levels of myc , elk‐1 , smad4 , hspb1 , tp53 , fadd , casp3 , and cflar . Next, microarray analysis will be performed to investigate the changes in gene expression on the human transcriptome. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .