Inducing Proliferation and Cell Survival of SW620 Colon Cancer Cells

Abstract

Vitamin C, also known as ascorbic acid, has been used for treatments for all kinds of cancers because it is known to stop the proliferation of cancer cells. For this experiment, we tested SW620 colon cancer cells by treating the cells with 1.5 mM of Vitamin C. We then analyzed these colon cancer cells by Trypan Blue, Propidium Iodine through the flow cytometry and also the WST-1 assay analysis and Caspase 3/7 analysis. The results showed unusable data because the cell viability of the treated cells decreased as did the non treated cells which disproves our hypothesis.

This experiment did not provide the right results needed to prove the hypothesis and requires more experimination and more variety of the concentration of vitamin C in order to obtain usable data.

Introduction

Cancer remains a feared and dreaded disease in the modern era. According to World Health Organization (WHO) global statistics, cancer is the second main cause of death. In 2018, it was estimated that around 9.

6 million people worldwide died due to cancer (Ghosh, et al., 2019). There has been many controversies saying the Vitamin C can be used to fight off cancer. Many scientists have studied Vitamin C and how it can be used to fight off this disease. For example, the clinical trials in a study done by Cameron & Pauling has shown when 100 terminal cancer patients were given supplement ascorbate, the results showed that the survival rate was 4.2% greater than those patients who were not given supplements. Another study done by Zeng has shown that when patients were given 2mM of Vitamin C, it suppressed the cell migration and invasion of the breast cancer cells.

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This can help prove that Vitamin C can be used to suppress the cancer cell. There are other studies regarding how Vitamin C can stop cancer cell progression; a study done by Satheesh has shown that Vitamin C induces its effect via interfering with the energy metabolism or cancer epigenome regulation in cancer stem cells. Another study by Gan did a test of vitamin C with breast cancer cells, and the results showed that vitamin C inhibits TNBC metastasis by affecting the expression of SYNPO2 and YAP1. Vitamin C is an essential micronutrient for humans and needs to be consumed through a Vitamin C-rich diet or supplements. Vitamin C is being used for treatments such as colon cancer.

According to the American Cancer Society, colon cancer is the third most common cancer diagnosed in both men and women in the United States. The American Cancer Society estimates the number of colorectal cancer cases in the United States for 2020 is 104,610 new cases of colon cancer. In the United States, colorectal cancer is the third leading cause of cancer-related deaths in men and in women, and the second most common cause of cancer deaths when men and women are combined. Colorectal cancer is a cancer that starts in the colon or the rectum. According to Ivanov, transformation of colon epithelial cells results from the activation of oncogenes as well as the inactivation of tumor suppressor genes controlling cell proliferation and survival. In colon cancer, cell lines with microsatellite instability (MSI) the high overall mutation frequency caused by the inactivation of DNA mismatch repair (MMR) genes results in the mutational inactivation of tumor suppressor genes.

With this knowledge about colon cancer, we can show that Vitamin C can help the suppression of cancer cells. Vitamin C can enter the colon cancer cell through the SVCT, which are sodium dependent Vitamin C transporters. Vitamin C at physiological concentrations, combined with hypomethylating agents may act synergistically to cause DNA demethylation, the process of the removal of a methyl group from cytosines, which can fight off the cancer cell. This experiment needs to have a positive and a negative control. In the negative control we will be using will be penicillin at 6%. The reason that the number 6% was chosen is because according to Banerjee, penicillin at 10%, 7.5%, and 5% was able to kill a cancer cell within 24 hours.

Another example is from the study by Blank; in this study he showed that it decreases cell proliferation in melanoma cells. The 6% penicillin was determined so the cancer cells do not die too quickly and that we are able to examine the cancer cells when they are dying. The positive control is a serum because the serum stimulates the Na+-pump-mediated uptake of K+ causing more cells to proliferate. The hypothesis of this experiment is that SW620 colon cancer cells treated with 1.5 mM with vitamin C will have reduced cell proliferation and cell survival. The hypothesis will be tested through the Trypan Blue Analysis, Cell Cycle and Viability Analysis with Propidium Iodide Staining, and Colorimetric Cell Proliferation of WST-1 assay and Caspase 3/7 assay.

Material and methods

For this experiment, SW620 colon cancer cells were used to see if the vitamin will cause the cancer cells to proliferate and survive. The positive control that was used was 20% serum, and the negative control was 6% penicillin antibiotic. Vitamin C at 1.5mM was used throughout the four parts of the experiment. The cells were incubated before each test and assay to ensure the best result possible. These plates had one positive control of the 20% serum, 1 negative control of the 6% penicillin, three treated wells, and 3 non treated wells. The WST-1 and the Caspase 3/7 assay had a 96-well plate with a density of 1×10^4. These plates had one positive control which was the 20% serum, 1 negative control which was the 6% penicillin, three treated wells, and 3 non treated wells.

Trypan Blue Exclusion Test

For the Trypan Blue Exclusion Test, we used a 24-well plate with a density of 2×10^5. The wells consisted of 3 untreated wells containing the SW620 cells, 3 wells containing 1.5 mM Vitamin C treated cells, 1 well containing the 20% serum positive control, and 1 well containing the 6% negative control. The cells were incubated for 2 days at 37 degree C. After incubation, the cells were transferred to a new vial and washed down with 250 ul sterile PBS. After washing the cells with PBS, the PBS was removed and 150 ul of Trypsin 25% solution and 150 ul the growth media was added to all of the wells. We mixed the solutions and transferred them to labeled sterile microcentrifuge tubes. Cells were centrifuged at 1500 rpm for five minutes. After centrifuging, the cells became pellets which were then isolated for each tube then resuspended by adding growth media and pipetting it up and down. After resuspending the pellet, the cell pellets were then placed into new sterile tubes that have 10 ul 0.4% trypan blue solution. Then 10 ul of the cells were extracted and placed onto the hemocytometer to analyze and record total cell count and cell viability.

Cell Cycle and Viability Analysis by Propidium Iodide Staining

For the Propidium Iodide Staining Test, the 24-well plate had a density of 2×10^5 cells with a growth medium. Similar to the Trypan Blue Exclusion test, the wells consisted of 3 wells of Sw620 cells, 3 vitamin treated wells, one positive control, and one negative control. The incubation for the cells was for 48 hours at 37 degree C. After incubation, growth media was removed and the cells were washed with 1000 ul of sterile PBS. We then removed the PBS and added 500 ul 0.25% trypsin solution and incubated the plate at room temperature. During incubation the plate needs to be tapped every three minutes so that the cells can attach. After the incubation period and the cells have attached, the growth media was added and transferred to clean, sterile, and labeled centrifuge tubes.

The tubes were placed into a centrifuge and centrifuge for 5 minutes at 1000 rpm. After centrifuge, the growth media was removed and sterile PBS was added to wash the cell. The tubes were divided into two sets, one for cell viability and one for cell analysis. For cell viability, 0.5 ul of PI was added and then looked under the cytometer for analysis. For cell cycle analysis, PI and 100% ethanol was added dropwise while vortexing the tube. The tubes were parafield and left to incubate for 1 hour at 4 degree C. After incubation, the tubes were centrifuged for 3 minutes 1500 rpm. After the centrifugation, the ethanol was extracted and the solution was washed with sterile PBS. The tubes were incubated in the dark for 40 minutes. The cells were resuspended in Sterile PBS and analyzed on a flow cytometer.

Colorimetric Cell Proliferation (WST-1) Assay

The cells were placed in a 24- well plate at a density of 1 x 10^4 cells/wells in 0.1 ml of complete growth medium. The plate had 3 wells of control, 3 wells of vitamin treated cells, 3 wells of negative control, and 3 wells of positive control. Incubate the cells in the 37 degree C incubator for two days and add 10 ul of WST-1 mixture to each well. Pipette up and down several times to mix well. Incubate the plate at 37 degree C for 2 hours and then measure the absorbance of each well at 450 nm using a microplate reader.

Caspase 3/7 Assay

The cells were placed in a 96-well plate at a density of 1 x 10^4 cells/wells in 0.1ml of complete growth medium with or without vitamins. The plate had 3 wells for the control, 3 wells for vitamin treated cells, 3 wells for the negative control, and 3 wells for the positive control. Incubate the cells in the 37 degree C incubator for two days and then add 100 ul/well (96-well plate) of caspase 3/7 working solution. Incubate the plate at room temperature for at least 1 hour, protected from the light. Measure the absorbance of each well 490nm using a microplate reader.

Results

The results that came from the trypan blue test helped analyze the amount of total cells for each well. For the control group, the C2 well was an outlier, because we added the wrong amount of trypsin solution; it showed a result that invalid for this experiment (Figure 2). Excluding the outlier, the average total cell count in the control groups had 50% less total cells than the positive control. The average total cells in the vitamin treated group has more than 50% more cells than the negative control (Figure 1). The positive control did show 70% more total cells than the negative control, as shown in Figure 2. The results for the cell viability showed the average for the controlled groups was at 99.4%, meanwhile the average cell viability for the negative control groups was 96.4%.

The Propidium Iodide (PI) test showed the amount of viable cells in the cell cycle analysis and the cell viability throughout the flow cytometry. The Trypan Blue Test analyzed the amount of total cells in each well (Figure 1). The average amount of total cells in the control groups (C1-C3), ignoring the outlier, had 50% less total cells than the positive control. The average total cells in the vitamin treated group (V1-V3) has 52% more cells than the negative control. Comparatively, the positive control displayed 70.5% more total cells than the negative control. Figure 2 also displays significance of an average 99.4% cell viability in the control groups, compared to an average 96.5% cell viability in the vitamin treated groups .

The Propidium Iodide (PI) Test calculated the amount of viable cells in cell cycle analysis and cell viability through flow cytometry. Figure 3 displays 84.1% more viable cells in average control groups than the average vitamin treated groups. Percentage of viable cells in G0/G1 and G2/M phases of each group (figure 4) displayed greater percentages of viable cells in the G0/G1 phase compared to the G2/M of each group. The greatest percentage of viable cells was 58.37% in the positive control of the G0/G1 phase, where the lowest percentage of viable cells was 15.91% in control group 1 (C1) in the G2/M phases.

The Propidium Iodide Test, where fluorescent dye stains the DNA of non-viable cells, was tested through flow cytometry to calculate the amount of viable cells in the cell cycle analysis and cell viability. For cell viability, the controlled groups in Figure 3 shows that 84% are more viable in the control groups than the average vitamin treated groups in the cell cycle analysis. The data for the G0/G1 phase and G2/M phases shows that G0/G1 phase of the control groups have greater cell viability percentages than the vitamin control groups. Looking at the percentages for the cell viability, the data shows that there is no distinct relationship between the control groups and the vitamin control groups.

The Propidium Iodide Test shows 12,000 viable cells in all 8 groups that were tested. The cell count was capped at 12,000, but there were likely more cells present. The percentages of the cells in Figure 5 are for the Propidium Iodide Negative (PI-N) and Propidium Iodide Positive (PI-P) for the 8 groups that were tested. The PI-N data represented the viable cells in the wells, while PI-P data represented non-viable cells. Figure 5 shows the V2 outlier, but it was excluded in calculations. Excluding the outlier, data shows that there were 1.7% more average viable cells in the vitamin treated than the control groups. Figure 5 was able to determine a relationship between PI-N and PI-P because there was a smaller percentage of the PI-P in vitamin treatment.

The WST-1 analysis and Caspase 3/7 analysis were used to calculate the P-Values we obtained. The P-values show the likelihood of values from Tables 3 and 4 were due to chance, being that significant is 0.05. For the Anova Single Factor calculation, the WST-1 and the Caspase 3/7 are less than the 0.05 indicating significance. The T-test values reveal there is a significance in the WST-1 Assay but not Caspase 3.7 Assay. The data was able to determine that there was a 2.6% decrease in survival of cells in the vitamin treated well than untreated wells. The WST-1 Assay in Table 4 shows the average number of living cells in WST-1 at the absorbance reading of 450nm. Figure 4 shows that there were 10.1% less living cells in the vitamin treated group than the untreated group.

This assay also shows a decrease in cells in the vitamin treated group. In Table 4, there are 10.1% less living cells in the vitamin treated group. The hypothesis states that SW620 colon cancer cells treated with 1.5 mM with vitamin C will have reduced cell proliferation and cell survival. The results from the Trypan Blue, Propidium Iodine, the WST-1 assay analysis and Caspase 3/7 analysis did not show the results we wanted. The results showed too much variation that we could not support or reject our hypothesis. In the Trypan Blue Exclusion Test, we saw that the trypan blue did not penetrate the membrane of the dead cells under the microscope.

Looking at the results for the Trypan Blue test, the data we received did not support the hypothesis because there were more total cells in the vitamin treated cells than the untreated cells. We had an outlier in Figure 2, due to not properly removing all of the supernatant. However in Figure 1, the data shows that the vitamin treated cells have less cells than the controlled. With these data, it is hard to determine if our hypothesis is correct or not due to the variation of these two graphs.

When doing the PI test, the hypothesis predicts that there will be less cells in the untreated G0/G1 phase than the G2/M phase. In Figure 4, it was shown that the viability of the vitamin treated cells in the were less than the untreated cells, but there were more cells in the G0/G1 phase because it’s the start of the cell cycle, which is prone to have more cells. In the G2/M phase, the cells divide, so it is prone to have less cells, but the data we have did not show a trend between vitamin treated and untreated. Figure 5 did not prove our hypothesis because the data we obtained showed that the vitamin treated cells have more PI-N cells than the untreated. This data showed that there were viable cells in the vitamin treated wells than the untreated wells. This test has provided us with data that neither supports or rejects our hypothesis because there is too much variation in cell count and viability.

The results of this experiment cannot definitively prove or disprove our hypothesis. The data shows too much variation in showing if the cells in the vitamin treated wells have more cells than the untreated wells. Some data proved our hypothesis and other data completely rejected our hypothesis. Further time and research is required in order to determine if this hypothesis is right or not. Vitamin C can be a good method in order to fight off cancer but we need further experiments to solidify that Vitamin C can stop the proliferation of SW-620 colon c