Project Title

15 Lox 1 Up-regulation and Cytotoxicity with γ-tocotrienol in HCT-116 Colon Cancer Cells

Authors' Affiliations

Lindsey C. Shipley, BS1; Harika Balagoni, MD2; Janet Lightner3; Victoria Palau, PhD4; Krishnan Koyamangalath, MD3 1East Tennessee State University, Quillen College of Medicine 2East Tennessee State University, Division of Internal Medicine 3East Tennessee State University, Division of Hematology & Oncology 4East Tennessee State University, Division of Pharmacology

Location

Mt. Mitchell Room 220

Start Date

4-5-2018 8:00 AM

End Date

4-5-2018 12:00 PM

Poster Number

130

Name of Project's Faculty Sponsor

Dr. Krishnan

Faculty Sponsor's Department

Hematology and Oncology

Type

Poster: Competitive

Classification of First Author

Medical Student

Project's Category

Biomedical and Health Sciences

Abstract Text

Colorectal cancer is the second leading cause of cancer-related deaths in the United States and the third most common cancer in men and women. Vitamin E is a lipid soluble antioxidant that exists as eight structurally different isoforms of tocopherols and tocotrienols. Recent experimental, and molecular studies suggest that γ-tocotrienol (GT3) may be a more potent cancer-preventive form of vitamin E. 15-lipoxygenase-1 (15-LOX-1) and its product 13-S-hydroxyoctadecadienoic acid (13-S-HODE) are decreased in colon cancer cells. 15 LOX-1 is considered a tumor suppressor gene in colon carcinogenesis. Non-steroidal anti-inflammatory drug (NSAID)-induced 15-LOX-1 expression is critical to aspirin and NSAID-induced apoptosis in colorectal cancer cells. HCT-116 is a microsatellite-instability (MSI) colon cancer cell line. MSI is a marker of chemo-resistance but is associated with improved survival as compared to microsatellite-stable (MSS) colon cancers. The effects of GT3 on cytotoxicity and 15 LOX-1 expression was studied on the human colon cancer cell line HCT-116. HCT-116 colon cancer cell lines were cultured in DMEM media and dosed with increasing concentrations of GT3 (20µM-50µM). Cytotoxicity of the drugs was studied using Cell Titer Glo and MTS assays 24 hours after dosing. Cells were then plated in 6-well plates and grown for 24 hours. Cells were then dosed with 2 mL of GT3 at 20 uM at the respective time periods (2h, 4h, 6h, 12h, 16h, 24h) and lysates were harvested. Gel electrophoresis was run according to BCA protein assay from the time-dependent lysates and blots were tagged with a rabbit 15-lox antibody. Ongoing experiments include RNA PCR. RNA is being isolated at 2, 4, 6 and 12 hours. The RNA as reversed transcribed using a 15 lox 1 primer and that cDNA is being quantified using Quantitative PCR. GT3 induced cytotoxicity in MTS assay and Cell Titer Glo assay when added to HCT-116 cell line. 15 LOX 1 protein expression was found to be up-regulated in the colon cancer cell line HCT-116 when GT3 was added at 12h, 16h and 24h with the maximum expression at 16 hours. Chemotherapeutic drugs can have significant side effects. Understanding the role of GT3 on colon cancer cell lines could lead to the development of novel drugs to supplement current chemotherapy regimens and allow for lower doses of chemotherapeutic agents. Modulation of 15-LOX-1 suggests that GT3 may induce apoptosis through induction of the lipoxygenase pathway. Further experiments are under way to study the mechanism of action of GT3 on the 15 LOX-1 pathway. Since HCT-116 is a MSI- colon cancer cell line, effects of GT3 on MSS- colon cancer cell lines will also be studied.

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Apr 5th, 8:00 AM Apr 5th, 12:00 PM

15 Lox 1 Up-regulation and Cytotoxicity with γ-tocotrienol in HCT-116 Colon Cancer Cells

Mt. Mitchell Room 220

Colorectal cancer is the second leading cause of cancer-related deaths in the United States and the third most common cancer in men and women. Vitamin E is a lipid soluble antioxidant that exists as eight structurally different isoforms of tocopherols and tocotrienols. Recent experimental, and molecular studies suggest that γ-tocotrienol (GT3) may be a more potent cancer-preventive form of vitamin E. 15-lipoxygenase-1 (15-LOX-1) and its product 13-S-hydroxyoctadecadienoic acid (13-S-HODE) are decreased in colon cancer cells. 15 LOX-1 is considered a tumor suppressor gene in colon carcinogenesis. Non-steroidal anti-inflammatory drug (NSAID)-induced 15-LOX-1 expression is critical to aspirin and NSAID-induced apoptosis in colorectal cancer cells. HCT-116 is a microsatellite-instability (MSI) colon cancer cell line. MSI is a marker of chemo-resistance but is associated with improved survival as compared to microsatellite-stable (MSS) colon cancers. The effects of GT3 on cytotoxicity and 15 LOX-1 expression was studied on the human colon cancer cell line HCT-116. HCT-116 colon cancer cell lines were cultured in DMEM media and dosed with increasing concentrations of GT3 (20µM-50µM). Cytotoxicity of the drugs was studied using Cell Titer Glo and MTS assays 24 hours after dosing. Cells were then plated in 6-well plates and grown for 24 hours. Cells were then dosed with 2 mL of GT3 at 20 uM at the respective time periods (2h, 4h, 6h, 12h, 16h, 24h) and lysates were harvested. Gel electrophoresis was run according to BCA protein assay from the time-dependent lysates and blots were tagged with a rabbit 15-lox antibody. Ongoing experiments include RNA PCR. RNA is being isolated at 2, 4, 6 and 12 hours. The RNA as reversed transcribed using a 15 lox 1 primer and that cDNA is being quantified using Quantitative PCR. GT3 induced cytotoxicity in MTS assay and Cell Titer Glo assay when added to HCT-116 cell line. 15 LOX 1 protein expression was found to be up-regulated in the colon cancer cell line HCT-116 when GT3 was added at 12h, 16h and 24h with the maximum expression at 16 hours. Chemotherapeutic drugs can have significant side effects. Understanding the role of GT3 on colon cancer cell lines could lead to the development of novel drugs to supplement current chemotherapy regimens and allow for lower doses of chemotherapeutic agents. Modulation of 15-LOX-1 suggests that GT3 may induce apoptosis through induction of the lipoxygenase pathway. Further experiments are under way to study the mechanism of action of GT3 on the 15 LOX-1 pathway. Since HCT-116 is a MSI- colon cancer cell line, effects of GT3 on MSS- colon cancer cell lines will also be studied.