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The A375 cell line, also known as malignant melanoma, is a type of skin cancer. This investigation was undertaken because there was a need to establish an in vitro experiment that recapitulated malignant melanoma to evaluate the therapeutic potential of L-ascorbic acid. The ascorbic acid treatment has anticancer qualities, therefore it can stop the cell cycle and/or cause cell death. In order to see the effect of L-ascorbic acid in vitro, a portion of the A375 cells were treated with 10µL of the 1500µM L-Ascorbic acid dilution and 990µL of IMDM (1X); however, the rest of the A375 cells were treated with 10µL of Sterile Deionized Water and 990 µL of IMDM (1X).
The results of the investigation show evidence that there is a decrease in A375 cells once the L-ascorbic acid treatment is applied. It was concluded that L-ascorbic acid has the capabilities of curing cancer in vitro.
It is vital to have sufficient amounts of ascorbic acid (Vitamin C) in our body, due to its specific properties.
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Ascorbic acid cannot be produced or stored by humans and must be attained by diet (National Center for Biotechnology Information). Ascorbic acid helps the metabolism of tyrosine, tryptophan, and folic acid; it decreases cholesterol levels; it helps the body to absorb and breakdown histamine; it promotes the synthesis of collagen; it neutralizes free radicals (no unpaired valence electron); it secures the DNA from damage due to mutagens and free radicals; it decreases the risk of premature death; it fights off widespread environmental pollutants (Mastarangelo et al, 2017).
Therefore it is evident that vitamin C helps multicellular animals, such as humans, stay healthy. More importantly, ascorbic acid can fight specific diseases such as cancer.
Malignant Melanoma (A375 cell line) is a significant type of skin cancer that begins in the pigmentation system on the skin (Jung-Feng Hsieh et al, 2011). Recent studies showed that high-dose ascorbic acid have cytotoxic effects on cancer cells; however, ascorbic acid only shows anticancer properties at specific dosages. However, in human metastatic melanoma cells, only pharmacological doses of ascorbic acid promote significant change in histone acetylation and the DNA methylation (Venturelli et al, 2015). Therefore, high-doses of vitamin C change expression of genes.
In human A375 melanoma cells, the ascorbic acid treatment can stop the cell cycle and/or cause cell death (Venturelli et al, 2015) To stop the cell cycle and/or cause apoptosis, L-ascorbic acid has its own mechanism. The L-ascorbic acid is responsible for two main properties. First, it regulates redox reactions, which regulate H2O2 generation (oxidative cell damage) and oxidative damage due to glutathione depletion. Second, it regulates co-factor for 2-OG dioxygenases, which is responsible for the upregulation for both epigenetic demethylases and HIF hydroxylases (Vissers & Das, 2018).
As previously mentioned, cancer cells make high quantities of H2O2. High levels of H2O2 causes cell proliferation, change in DNA, metastasis, resistance to apoptosis, angiogenesis, and activation of the hypoxia-inducible factor 1 (HIF-1) (Mastarangelo et al, 2017). However, ascorbic acid inhibiting the HIF-1α transcriptional activity is what decreases levels of H2O2, which may change the malignant phenotype (Wang et al, 2018). It is clear that ascorbic acid directly affects the A375 cell line.
This investigation focused on the changes in the number of A375 cells once ascorbic acid was added in vitro. Given the work by previous scientists, this begs the question of what changes do A375 cell line go through when it is introduced to ascorbic acid at a concentration of 15mM. We hypothesized that when ascorbic acid is added to A375 cells, it will kill a fraction of the A375 cells due to its anticancer properties. Treatments like chemotherapy and radiation therapy can specifically target malignant melanoma and cure at times; however, there are side effects to these procedures. By doing further research we can come up with novel ways target malignant melanoma genes without harming normal cells. Our work evidently shows that there has been significant decrease in the number of A375 cells when treated with L-ascorbic acid.
The A375 cells were placed in the 12 well Corning CellBind Surface Polystyrene plate (Costar). 4 of the wells were used for the experimentals and 2 of the wells were used for the controls. The IMDM (1X) (Gibco) that was in the wells were aspirated. Then the malignant melanoma cells were washed with 100µL of Phosphate Buffered Saline (Dulbecco), but the PBS was aspirated immediately. The experimentals were treated with 10µL of the 1500µM dilution of L-Ascorbic acid (Sigma) and 990µL of IMDM (1X) (Gibco). The controls were treated with 10µL of Sterile Deionized Water and 990 µL of IMDM (1X) (Gibco). The plate was incubated at 37°C and at 5% CO2 for 18 hours.
When the 12 well plate was removed from the incubator, each well was aspirated and the cells were washed with 100µL of Phosphate Buffered Saline (Dulbecco), but the PBS was aspirated immediately. Then, 300µL of 0.05% Trypsin-EDTA (1X) (Gibco) was added to each well and the 12 well plate was incubated at 37°C and at 5% CO2 for 5 minutes. .When the plate was removed from the incubator, to the side was gently tapped in order to dislodge any adherent cells. 300µL of IMDM (1X) (Gibco) was added to each well then the solution in the wells were pipetted up and down in order to break up clumps of cells. 10 µL of Trypan Blue Solution 0.4% (w/v) in PBS was added to microcentrifuge tubes. Then, 10µL of the cell solution was transferred from each well to the microcentrifuge tubes. A coverslip was placed on a hemocytometer. 10µL of the Trypan Blue/cell solution was pipetted on the hemocytometer. Then the number of alive and dead cells were counted. This process repeated for each of the experimental and control wells.
The number of alive A375 cells after treatment. The experimental has an average alive cell count of 16.75 cells, and the control has an average alive cell count of 25 cells. The average number of dead cells in the control is higher than the experimental.
The number of dead A375 cells after treatment. The experimental has an average dead cell count of 17.57 cells, and the control has an average dead cell count of 6.5 cells. The average number of dead cells in the experimental is higher than the control.
The L-Ascorbic acid was introduced to a malignant melanoma cell line. The results show us that the experimental variables had a higher number of dead cells than the control had. It was hypothesized that ascorbic acid was going to kill a fraction of the A375 cells, due to ascorbic acid has anticancer properties. Such a hypothesis was carried out because the specific outcome of the mixture of ascorbic acid and the A375 cell line was unknown. It was expected that the A375 cancer cells were going to die, and the results came out to be consistent with the hypothesis. This experiment found that ascorbic acid has anticancer properties that fights against malignant melanoma.
The results our experiment put forward is confirmed by previous studies. Studies done by Tarumoto, Fromberg, Rouleau, and Yang state that when ascorbic acid is present at concentrations of 1 mM or more results in cell cycle arrest and/or cell death (Tarumoto et al., 2004; Fromberg et al, 2011; Rouleau et al, 2016; Yang et al, 2017). Also, in our study the data shows that the molecular mechanism has an active effect since the ascorbic acid treatment lowered the levels of H2O2 eliminating cell proliferation, change in DNA, metastasis.
The results of the investigation set forward an unexpected result, which was in the control variable. Sterile deionized water was added instead of the L-ascorbic acid to the control variable in order to observe the effectiveness of the treatment. However, the data shows that per well approximately there were 6.5 dead cells. The ideal number dead A375 cells is 0; however, there are reasons to why we did not have this cell count. The reasons to why we did not get the ideal number is because of study limitations. Those limitations can be cross contamination and the fact that some solutions that were used were expired.
Examining the Relationship Between Ascorbic Acid in A375 Cancer Cells. (2024, Feb 16). Retrieved from https://studymoose.com/document/examining-the-relationship-between-ascorbic-acid-in-a375-cancer-cells
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