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Biomedical Research

The Effects of Antioxidants on Mouse Osteoblastic Cancer Cells

Comparative Analysis of Effectiveness of Apoptosis

Abstract

As there are more than a million cases in the United States annually, and over 500,000 Americans die each year, cancer can be characterized as the continual unregulated rapid increase in the number of abnormal cells. The use of mouse model cancer cells serves an utmost significance in the field of research as the causal link to carcinogenesis, the initiation of cancer formation,  and cancer genes can be used to test and develop new therapies. Chemotherapy is the most common approach when looking at cancer treatment, where cancer cells are killed using chemicals that are toxic to living cells. Antioxidants maintain homeostasis and the maintenance of cell integrity in human immune systems and have shown to increase the life quality of the patients. The purpose of this experiment was to determine which antioxidant will be the most effective at different volumes to promote apoptosis in mouse osteoblastic cancer cells (MC3T3). The hypothesis was that as the volume of antioxidant solution increases, then the apoptosis of MC3T3 will increase. The experiment was conducted as MC3T3 cells were first grown for 24 hours, then the antioxidants Lycopene, Resveratrol, and Vitamin C were then added to the cells. The cells were once again grown for 24 hours, and after the 24 hours, cell viability was measured through MTT assay. The results show, the cell viability of Lycopene at 100uL was 27%, it was the most effective in promoting apoptosis. The hypothesis was supported to the extent that Lycopene and Resveratrol were successful in promoting apoptosis at certain volumes. This experiment can be applied to help cancer patients by offering another treatment or in combination with chemotherapy to increase the life quality of the patient.  

Variables/Purpose 

Purpose

The purpose of this experiment is to determine which antioxidant will be the most effective at different volumes of the antioxidant, to promote apoptosis in mouse osteoblastic cancer cells. 

Variables 

Independent Variable – The volumes of the different antioxidants that will be induced into the cells 

Dependent Variable – The cell viability of each antioxidant at different volumes, which will be measured using MTT assay 

Control – Micro-wells not being induced with any treatments 

Experimental Control – Etoposide, which is a chemotherapeutic agent proven to promote apoptosis and most prescribed chemotherapeutic agent in the world 

Introduction & Background Research 

Over 500,000 Americans die of cancer each year, as there are more than a million cases in the United States annually (Cooper, 2000). Cancer can be characterized as the continual unregulated rapid increase in the number of abnormal cells, leading to a group of cancer cells, which is a tumor (Cooper, 2000). Cancer is caused by carcinogens that start the growth of cancer cells; carcinogens are substances that promote or initiate the growth of cancer cells (Cooper, 2000). Cancer cells grow with continual proliferation, rather than healthy cells that respond appropriately to the signals that control normal cell behavior (Cooper, 2000). Tumors are a group of cancer cells that can be categorized into either benign or malignant (Cooper, 2000). A benign tumor is similar to a wart where it does not spread cancer to other tissues and other parts of the body and stays in a central location (Cooper, 2000). 

On the other hand, a malignant tumor is capable of spreading to other tissue as well another parts of the body through lymphatic and circulatory systems (Cooper, 2000). Among the various unique types of cancer, they are classified as either sarcoma, carcinoma, lymphomas, or leukemias (Cooper, 2000). Carcinomas, which are the most common, and occurs in 90% of human cancer cases, are malignancies of epithelial cells, which are sheets of cells that line internal organs and cover the surface of the body (Cooper, 2000). Sarcomas are tumors that are solid in connective tissues, like bones, cartilage, and muscle, which rarely occur in humans, and lymphoma or leukemia rarely occurs with 8% human cancer cases, comes from the cells of the immune system and blood-forming cells (Cooper, 2000). 

Metastasizing, which is the development of a malignant growth that is secondary to the primary site of cancer, which grows at a distance and spreads to other sites in the body (Macedo, Ladeira, Pinho, Saraiva, Bonito, Pinto, Goncalves, 2017). The third most frequent site of metastasis is the bone, which has a relative incidence percentage of 65-75% (Macedo et al., 2017). The three types of bone metastasis are classified as Osteolytic, Osteoblastic, or mixed (Macedo et al., 2017). Osteoblastic cancer is seen as the breakdown of the new bone similar to that of prostate cancer, small cell lung cancer, carcinoid, and Hodgkin lymphoma (Macedo et al., 2017). Bone metastasis is a sign that the disease has spread to other organs and predicts a short term probability of the disease in cancer patients (Macedo et al., 2017). Once cancer has spread to the bone, the chance of it being cured is low, though treatment can be applied to slow its growth because new drugs and treatments have shown a better quality of life and improved life expectancy (Macedo et al., 2017). Both Filipa Macedo and colleagues and Geoffrey Copper discuss the impact that cancer has left on society. Though they both discuss metastasis and how it leads to malignant tumors, Macedo looks at it only from bone metastasis, while Cooper analyzes metastasis through cancer in general. As they look at cancers through different outlooks, Macedo categorizes the four main types of cancers, and Coopers analyzes Sarcoma cancer, specifically bone cancer and the three types of bone cancers. 

When looking at metastasis it is also essential to look at carcinogenesis, which is how cancer formation starts, and neoplastic development, which is the formation of diseases that conditioned to cause tumor growth, which occurs when oxidative stress, damage from cancer cells, go unrepaired, that among humans and animals, research has shown the degree of biological and genetic similarity (Balmain & Harris, 2000). Mice develop tumors in the same tissues and amazingly also in the same histopathological course as humans even though they are much smaller animals with a higher metabolic rate (Balmain & Harris, 200).Humans have similar characteristics such as anatomical, cellular, and molecular to mice, which have the utmost  importance in function and critical properties in cancer (Tratar, Horvat, & Cemazar, 2018). The use of mouse models serves the utmost significance in the field of research as the causal link to carcinogenesis and cancer genes can be used to test and develop new therapies (Tratar et al. 2018). Both Balmain and colleagues and Tratar and colleagues discuss how mice cells are similar to human cells are similar biologically to the extent of their higher metabolic rate and other small factors. Tratar goes onto discuss how the use of mice cells can be useful in research to apply applications on to human cells. In contrast, Balmain looks more into the histopathology, examination of tissue at a microscopic level, similarities of the cell, such as the presence of Telomerase, an enzyme that helps maintain chromosome integrity in both human cells and mice cells. 

As mice cells serve as useful models of human cancer cells, the use of mice cells can be used to test new treatments such as chemotherapy drugs and antioxidants. Chemotherapy is the most common approach when looking at cancer treatment, where cancer cells are killed using chemicals that are toxic to living cells (Thyagarajan & Sahu, 2018). As chemotherapy has shown to be effective, it has many consequences such as DNA damage and damage to DNA replication, and it induces cell death into living healthy cells (Thyagarajan & Sahu, 2018). As these effects of chemotherapy affect the quality of life of cancer patients, there is a need for alternative treatment. Antioxidants maintain homeostasis and the maintenance of cell integrity in human immune systems (Pham-Huy & Lien Ai et al., 2008). Many different types of antioxidants can be found in vegetables; they have been shown to delay or prevent oxidative stress (Pham-Huy & Lien Ai et al., 2008). Antioxidants also serve as an effective treatment for cancer as studies have shown an increase in apoptosis, programmed cell death, in different cancer cell lines, and inhibit cell proliferation (Pham-Huy & Lien Ai et al., 2008). Both Pham-Huy and colleagues and Thyagaran and Sahu show the effectiveness of antioxidants as an anticancer treatment. Though Thyagaran and Sahu examine the relationship between the effectiveness of chemotherapy and its effects on antioxidants efficacy. Pham-Huy, in contrast to Thyagaran and Sahu, looks at specific antioxidants and their effectiveness in different clinical studies. As there are many different types of antioxidants, such as Lycopene, Resveratrol, and Vitamin C (ascorbic acid), it is crucial to examine their effectiveness against cancer cells and their effectiveness when compared to chemotherapeutic drugs such as Etoposide. 

Lycopene, which is mainly derived from tomatoes, tomato products, and other fruits, has shown to be a potent antioxidant and to reduce the incidence of cancer (Johary et al., 2012). Dietary carotenoids, a product of plants, such as Lycopene, have shown to be connected to the decreased risk of cancer and the maintenance of good health (Johary et al., 2012). Recent studies have shown Lycopene as an anticancer agent as its ability to inhibit metastasis, increasing the antioxidant response, preventing oxidative stress, and promoting apoptosis (van Breemen & Pajkovic, 2008). Lycopene has the potential, as a natural product, to be used in cancer treatment to increase the rate of apoptosis based on the studies that show Lycopene supplements when used against prostate cancer have shown reduced risk, anticancer activities, and chemoprevention, chemicals that prevent or slow development of cancer activities. Johary examines Lycopene as a dietary carotenoid and its many properties, which lead to beneficial effects such as preventing cancer. Van Breeman and Pajkovic look at the properties of Lycopene as an antioxidant and evaluates its efficacy by looking at clinical studies. Both authors view Lycopene from a different perspective, though they both establish it to be an effective antioxidant against cancer. 

Resveratrol is a product containing high levels of naturally occurring phenols (organic products), which is derived from plant sources, berries, peanuts, and grapes (Bishayee, 2009). Resveratrol has shown to be effective against the three stages of carcinogenesis: initiation, promotion, and progression (Bishayee, 2009) Resveratrol has a robust anticancer property as it can inhibit proliferation in human tumor cells (Ko et al., 2017). As Resveratrol can stop carcinogenesis, it has brought much attention to the prevention of cancer, a therapeutic drug, treatment researchers (Bishayee, 2009). Resveratrol can make cancer cells sensitive to chemotherapeutic agents and reverse multidrug resistance to cancer cells when used with drugs that are used clinically because this shows its efficiency in fighting cancer cells (Bishayee, 2009). Reports have shown Resveratrol can make cancer cells sensitive to chemotherapeutic agents and reverse multidrug resistance to cancer cells when used with drugs that are used clinically. Ko and colleagues examine Resveratrol specifically when used for cancer treatment and look into studies that inhibit Resveratrol into variety of cancers. Bishayee also examines resveratrol when used for cancer treatment, but specifically looks into Rodent studies which can further be implicated. Both Bishayee and Ko and colleagues see resveratrol as an effective anti-cancer treatment as they both discuss the effectiveness even looking at different types of cancers.

Ascorbic acid romote cell proliferation and cell differentiation in cancer cells (Yang & Seo, 2013). Ascorbic acid, also known as Vitamin C, in recent studies, has shown its ability to promote cell proliferation and cell differentiation in cancer cells (Yang & Seo, 2013). Ascorbic acid has also been shown to increase the osteogenic formation of bone in bone cancer cells when treated with cell differentiation (Yang & Seo, 2013). Vitamin C also has been shown to increase the survival rate when inhibited with organisms with cancer cells and increase the survival rate of the mice (Yeom et al., 2009). The administration of ascorbic acid in cancer patients when they were in the terminal stage showed the increase in the quality of life because of this experiment shows the sufficiency of ascorbic acid when induced in sarcoma cells (Yeom et al., 2009). 

Etoposide, an epipodophyllotoxin, a substance occurring naturally in Mayapple Plant, is structurally similar to another chemotherapeutic drug vincristine, a chemotherapeutic drug that is in the same group of drugs called alkaloids (Stadtmauer, 1989). Recent studies show the effectiveness of vincristine as an active agent against bone cancer, which is structurally similar to Etoposide (Stadtmauer, 1989). Higher doses of Etoposide will increase the rate of apoptosis in the cancer cells as shown to be an active agent when used at high dosages with other chemotherapeutic agents when applied to bone cancer (Stadtmauer, 1989). Etoposide has been shown to inhibit the growth of pancreatic cancer cells and induce apoptosis (Zhang & Zhang, 2013). Etoposide is a principal chemotherapeutic agent that has been shown to treat many types of cancers because, for more than two years, it remains the most prescribed drug for cancer treatment (Zhang & Zhang, 2013). 

Research on antioxidants and their effects on cancer cells have shown that higher dosages increase the rate of apoptosis. Though they all increase apoptosis when the dosage is increased, it is also essential to discuss which treatment would be the most effective in comparison to Etoposide, which is a known chemotherapeutic agent. A study that examines the effects of Lycopene, Resveratrol and Vitamin C (ascorbic acid) on mouse osteoblastic cells (MC3T3) would enable us to see which one is the most effective compared to Etoposide.

Hypothesis

As you increase the volume of the antioxidant solution in presence of mouse cancer cells, then the apoptosis of the mouse cancer cells will increase, because studies on antioxidants have shown to increase apoptosis in many different cancer cell lines.  

Methods/Materials

Mouse Osteoblastic Cancer cell line (MC3T3) and other special instruments were provided by the University of Arkansas Little Rock. Throughout the whole experimentation all equipment was provided sterile and goggles, aprons, gloves, and other lab safety precautions were taken. An incubator was used throughout the experiment and was constantly at 37 C with 5% CO2 atmosphere.

Subculturing

To first begin collecting cells from the original medium in the original mammalian cell culture flask, cells were washed with PBS (11.9 mM phosphates, pH 7.4, 13.7 mM NaCl, 2.7 mM KCl). Then using Trypsin-EDTA (0.05% Trypsin/0.53 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium), cells were dissociated and placed into a incubator for 5 minutes until cells detached. Then a centrifuge at 500 g for 5 minutes was used to separate both medium and cells. Cells were then resuspended into a 10 mL medium with 10% FBS which was supplemented with antibiotics penicillin 5 6 (500 units/ml) and streptomycin (500 units/ml). Cell density was then measured by staining cells with Trypan blue dye by using a hemocytometer. Cells were then seeded into a 96 well plate at 1.0 – 1.5 x 8000 cells per well with 100μl medium, where only 24 wells seeded. Cells were then left to be grown in the incubator for 24 hours.

1 A substance used to promote cell growth of microorganisms 2 Polyphosphate Buffer solution, used to maintain pH 3 is used to remove cells from original culture 4 A machine used to separate liquids or solids of different densities 5 Fetal Bovine Serum, used to increase within medium 6 Helps control bacterial contamination 

Treatment

Antioxidant solutions were prepared by either crushing Vitamin C pills or making solutions from gel tablets. 2 determinations of 10μl, 50μl, and 100μl of Vitamin C, Resveratrol, and Lycopene were made. Each antioxidant at each specific volume was placed into 2 wells. Etoposide at 10μl was also placed into wells. 2 wells were left with no treatment to serve as a control. Cells were then left to be grown in the incubator for 24 hours.

Cell Viability 

The method that was used to measure the cell viability in this experiment was MTT assay. MTT assay is the process by which analyzing the activity of mitochondrial-dehydrogenase as a way to the number of living cells. To first begin, the cells were washed with FBS twice that was medium free of any medium. After they were washed, 10μl of MTT solution (5 mg/ml) was added to the cell culture and then the microwell plate was left in the dark in the incubator for 4 hours because it is light sensitive. After the 4 hours, the medium was removed and the cells were lysed with dimethyl sulfoxide, which helps dissolve MTT formazan which was produced mitochondrial succinate dehydrogenase. The cells were then placed into a microplate cell reader at 570nm to measure the absorbance of the purple color of each well produced by the MTT solution. Determinations were performed in duplicates for each treatment for better statistical analysis. The results of MTT assay were presented as a % showing the control values obtained from untreated cells. The cell viability was calculated using the % of dead cells = (A sample – A blank) /(A control – A blank) x 100.

7 Helps control bacterial contamination 8 Dye used to identify living tissue or cells 9 To undergo Lysis, breaking down of cell membrane 

Data / Observations

  • Tables
TreatmentAbsorbance at 570 nm Trial #1Absorbance at 570 nm Trial #2
Control0.4050.344
10 μL Etoposide0.3650.298
Vitamin C (A)0.8021.017
Lycopene (A)0.5110.51
Resveratrol (A)0.130.101
Vitamin C (B)1.1831.053
Lycopene (B)0.3620.508
Resveratrol (B)0.2550.23
Vitamin C (C)1.561.691
Lycopene (C)0.1090.096
Resveratrol (C)0.4480.45
Table 1. Absorbance Readings at two different determination of antioxidants at various volumes
10μLof AntioxidantsAbsorbance at 570 nm
Control0.3745
10 μL Etoposide0.3315
Vitamin C (A)0.9095
Lycopene (A)0.5105
Resveratrol (A)0.1115
Table 2.. Effects of Antioxidants at 10uL on Cancer Viability. Shows the prominence of each antioxidants effect on the cancer cell culture
50μL of AntioxidantsAbsorbance at 570 nm
Control0.3745
10 μL Etoposide0.3315
Vitamin C (B)1.118
Lycopene (B)4.35
Resveratrol (B)0.2425
Table 3.. Effects of Antioxidants at 50uL on Cancer Viability. Shows the prominence of each antioxidants effect on the cancer cell culture.
100μL of AntioxidantsAbsorbance at 570 nm
Control0.3745
100 μL Etoposide0.3315
Vitamin C (C)3.251
Lycopene (C)0.1025
Resveratrol (C)0.898


Table 4.. Effects of Antioxidants at 100uL on Cancer Viability. Shows the prominence of each antioxidants effect on the cancer cell culture.

Graphs

Graph 1. Viability of Cancer Cells in 10uL of Antioxidant Solutions. Displays the different antioxidants’ effects on the cancer cell culture


Graph 2. Viability of Cancer Cells in 50uL of Antioxidant Solutions. Displays the different antioxidants’ effects on the cancer cell culture

Graph 3. Viability of Cancer Cells in 100uL of Antioxidant Solutions. Displays the different antioxidants’ effects on the cancer cell culture

Statistical Analysis

Table 4. ANOVA Test Results. Shows the statistical variance of data and since the f-value is lower than the p-value in this case, the data is not statistically varied. Since the p-value is higher than 0.05 (> 0.05) it is not statistically significant.

Photographs 

Figure 1. MC3T3 cells after being detached and added to medium

Figure 2. 96-well plate. Contains only the cancer cells and antioxidants before growth

Discussion 

The expected result of apoptosis increasing when increased volumes of antioxidant the solution was true to a minimal extent. The results of some of the antioxidants at specific volumes had the opposite effect, where the cell viability of MC3T3 increased significantly. The cell viability of Vitamin C among the three different volumes, increased 243%, 298%, and 868% respectively. This result could be due to the fact that at a certain point of volume of the antioxidant solution, the cells will then feed on the Vitamin C increasing the viability significantly. Fernandes and colleagues looked at Vitamin C at different volumes and saw a similar result that at cell viability, was increased in cells treated with low volume of Vitamin C with respect to untreated bone cancer cells. Etoposide did not increase apoptosis significantly though 89% of cell viability was present after 10μL was seeded. Some antioxidants at specific volume were more effective in promoting apoptosis than the Etoposide. Lycopene at 100μL was the most effective in promoting apoptosis as cell viability was 27%, though at volume of 10μL and 50μL, it was 136% and 1162%. .The same effect that was on Vitamin C can be seen on Resveratrol as at a certain volume, the volume of the antioxidant will feed the cells until a certain volume has reached. For resveratrol, the opposite effect can be seen as at a lower volume of 10μL the cell viability was the lowest and as volume was increased the cell viability exponentially An ANOVA test was performed, and it ended with a p-value of 0.58602 and an f-value of .55892. This signifies that the data has an extreme variance between each treatment since they all had different results. Since the p-value is above 0.05, we have failed to reject the null hypothesis which is that there is no correlation between antioxidant treatment and cell viability. Within the experiment, there may have been some possible error as the vitamin C solution turned the well yellow while every other well had a more purple color. This may have been related to the vitamin’s natural color, but it could have influenced the results of the experiment. Due to time constraints, only 2 determinations were performed. Contamination could have occured as cells were being grown in an incubator with other organisms, or light contamination as MTT assay has to be done in a dark environment. 

Conclusion 

The hypothesis that as you increase the volume of the antioxidant solution in presence of mouse cancer cells, then the apoptosis of the mouse cancer cells will increase, was supported to the extent at which Resveratrol and Lycopene increased apoptosis when induced. Each antioxidant significantly demoted growth to a certain extent within the cancer cell apart from the Vitamin C solution. Furthermore, the polychemotherapy can be investigated by looking at not only chemotherapy but as well as a combined treatment of antioxidants and chemotherapy. Research can also be done on looking at the patient’s quality of life as they are being given normal chemotherapy and to also see if it is different as antioxidants are introduced. 

Acknowledgments

This project required an immense amount of outside help from Dr. Nawab Ali and Mrs. Puja. The supervision of a qualified scientist of her expertise was necessary in order to follow procedures accordingly. Dr. Ali also gave great advice throughout the process. Without the assets and resources provided by the Dr. Ali’s lab, there would have been no way for this project to come about. Mrs. Becker and Mrs. Norris contributed a significant amount of help throughout the research and statistical analysis process.

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Saad Shabbir, Youth Medical Journal 2020