Sulindac Sulfide’s Apoptotic and Anti-proliferative Effects on Human Colon Cancer Cells
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Non-steroidal anti-inflammatory drugs (NSAIDs) have been proven to decrease the incidence and mortality of colon cancer (11). One specific NSAID that has garnered much intrigue for its exceptional ability to prevent precancerous lesions and to act as a chemotherapeutic is sulindac. The purpose of our study was to evaluate a novel anti-proliferative mechanism of sulindac sulfide (SS) in colon cancer cells. We hypothesized that sulindac mediated its apoptotic effect through the inhibition of the p65 subunit of NF-KB resulting in increased p53 stability and ultimately cell cycle arrest/cell apoptosis. In order to simulate sulindac’s effect on colon cancer cells, we treated three colon cancer cell lines (HCT- 116 wild-type (WT), HCT-116 p53 -/-, and HT-29) with SS to observe the effects on proliferation, apoptosis, and the proteins that influence cell cycle. First, we evaluated the effect of SS on colon cell proliferation using HCT116 p53 WT cells; we observed a significant decrease in cell growth at the 48 and 72 hour time points. Next, we evaluated the protein expression of transcription factors known to regulate cell cycle progression and cell growth. HCT-116 WT cells were treated with 50 μM of SS for 24, 36, and 48 hours, lysed and used for western blotting. The transcription factor E2F1 was downregulated in the presence of SS starting at 24 hours; but its downstream target, dihydrofolate reductase (DHFR), is not inhibited until 48 hours of treatment. Based on these results we decided to evaluate if the observed decrease in proliferation was due to cell cycle arrest. Next, we stained the three colon cancer cells with Propidium iodide, a commonly used dye to quantify DNA content in cells when using flow cytometry, we evaluated the effect of SS (50uM) on cell cycle progression of HCT-116 WT, HCT-116 p53-/- and HT-29 colon cancer cell lines. With our results, we concluded that there was no significant difference in cell cycle observed between the control and treated tests of each respective cell line. To confirm these results we evaluated the protein expression of the cyclin kinase inhibitor, p21, which is known to function as a regulator of cell cycle progression at G1 and S phase. Based on our western blot results we determined SS does not regulate the expression of p21. Previous literature has stated that NSAIDs use reactive oxygen species (ROS) in mediating their cytotoxic effects. To evaluate if this was the potential mechanism of SS in colon cancer, we treated cells with SS followed by dichlorodihydrofluorescein diacetate (H2 DCFDA) and used flow cytometry to evaluate the reduction of this compound. In the SS treated cells there was an increased presence of reactive oxygen species (ROS) present. Based on the known interaction of ROS and NF-κB we decided to evaluate if SS mediated its function through the regulation of NF-KB. We determined that SS treatment decreases the nuclear translocation of the p65 subunit of NF-κB but has no effect on the presence of the phosphorylated/activated p65 subunit. We evaluated the effect of SS treatment of cell apoptosis induction using flow cytometry analysis of Annexin V and PI staining and we found that the HCT-116 WT cells had an increase in apotptosis while the other two cell lines experienced no significant difference in apoptosis. In conclusion, SS is most effective in inducing apoptosis of cells with a normal expression of the p53 protein due to an increase in ROS and may be an effective method for the treatment of cancers with a normal p53 protein due to its induction of apoptosis.
Recommended Citation
Martin, Samuel D., "Sulindac Sulfide’s Apoptotic and Anti-proliferative Effects on Human Colon Cancer Cells" (2019). Summer Research Internship Program. 23.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/23
Sulindac Sulfide’s Apoptotic and Anti-proliferative Effects on Human Colon Cancer Cells
LSU Health Medical Education Building
Non-steroidal anti-inflammatory drugs (NSAIDs) have been proven to decrease the incidence and mortality of colon cancer (11). One specific NSAID that has garnered much intrigue for its exceptional ability to prevent precancerous lesions and to act as a chemotherapeutic is sulindac. The purpose of our study was to evaluate a novel anti-proliferative mechanism of sulindac sulfide (SS) in colon cancer cells. We hypothesized that sulindac mediated its apoptotic effect through the inhibition of the p65 subunit of NF-KB resulting in increased p53 stability and ultimately cell cycle arrest/cell apoptosis. In order to simulate sulindac’s effect on colon cancer cells, we treated three colon cancer cell lines (HCT- 116 wild-type (WT), HCT-116 p53 -/-, and HT-29) with SS to observe the effects on proliferation, apoptosis, and the proteins that influence cell cycle. First, we evaluated the effect of SS on colon cell proliferation using HCT116 p53 WT cells; we observed a significant decrease in cell growth at the 48 and 72 hour time points. Next, we evaluated the protein expression of transcription factors known to regulate cell cycle progression and cell growth. HCT-116 WT cells were treated with 50 μM of SS for 24, 36, and 48 hours, lysed and used for western blotting. The transcription factor E2F1 was downregulated in the presence of SS starting at 24 hours; but its downstream target, dihydrofolate reductase (DHFR), is not inhibited until 48 hours of treatment. Based on these results we decided to evaluate if the observed decrease in proliferation was due to cell cycle arrest. Next, we stained the three colon cancer cells with Propidium iodide, a commonly used dye to quantify DNA content in cells when using flow cytometry, we evaluated the effect of SS (50uM) on cell cycle progression of HCT-116 WT, HCT-116 p53-/- and HT-29 colon cancer cell lines. With our results, we concluded that there was no significant difference in cell cycle observed between the control and treated tests of each respective cell line. To confirm these results we evaluated the protein expression of the cyclin kinase inhibitor, p21, which is known to function as a regulator of cell cycle progression at G1 and S phase. Based on our western blot results we determined SS does not regulate the expression of p21. Previous literature has stated that NSAIDs use reactive oxygen species (ROS) in mediating their cytotoxic effects. To evaluate if this was the potential mechanism of SS in colon cancer, we treated cells with SS followed by dichlorodihydrofluorescein diacetate (H2 DCFDA) and used flow cytometry to evaluate the reduction of this compound. In the SS treated cells there was an increased presence of reactive oxygen species (ROS) present. Based on the known interaction of ROS and NF-κB we decided to evaluate if SS mediated its function through the regulation of NF-KB. We determined that SS treatment decreases the nuclear translocation of the p65 subunit of NF-κB but has no effect on the presence of the phosphorylated/activated p65 subunit. We evaluated the effect of SS treatment of cell apoptosis induction using flow cytometry analysis of Annexin V and PI staining and we found that the HCT-116 WT cells had an increase in apotptosis while the other two cell lines experienced no significant difference in apoptosis. In conclusion, SS is most effective in inducing apoptosis of cells with a normal expression of the p53 protein due to an increase in ROS and may be an effective method for the treatment of cancers with a normal p53 protein due to its induction of apoptosis.
Comments
Mentors: Ches’Nique Phillips and Yaguang Xi