Author: Ashley Thomas Uppani
Peer Reviewer: Paula Zhu
Professional Reviewer: Robert Helm
Gemcitabine is a nucleoside analogue utilized in chemotherapy against various cancers. Despite its extensive anticancer activity against solid tumors and resulting cancer cell apoptosis, the inadvertent destruction by gemcitabine of healthy cells neighboring tumors decelerates the healing process and presents detriments. This research project examines the cytotoxic effect of gemcitabine, and how N-acetylcysteine (NAC) antioxidant can abate oxidative stress on cellular membranes, lessening cytotoxicity when used with gemcitabine. It was hypothesized NAC use in addition to gemcitabine treatment will decrease cell cytotoxicity, a consequence of oxidative stress, and prevent cell death.
Two separate experiments, pot plating and liquid/turbidity test, were conducted. Both experiments utilized the same concentrations of Yeast-Extract Peptone Dextrose media and gemcitabine with varying NAC concentrations in order to analyze NAC’S effect on yeast cell growth. Spot plating was the visual representation of NAC influence on yeast cell growth in various conditions over one day. Compared to the control dish, it was clearly visible that gemcitabine killed the yeast cells (Dish 2). Increasing concentrations of NAC allowed more cell survival comparable to the control. At the highest concentration tested of 800 µL, NAC was most effective in rescuing yeast cells in spot plating with a 100 µL concentration of gemcitabine. The liquid media graph showed the growth trend (OD600) of the yeast cells over eight hours. The highest tested NAC concentrations of 120 µL and gemcitabine concentrations of 60 µL were most effective in reinforcing yeast cell survival during liquid media tests. Therefore, since gemcitabine cytotoxicity is due to oxidative stress on cell membranes, and the results show NAC effectiveness at decreasing the cytotoxicity side effect of gemcitabine, the addition of NAC would allow more efficacious cancer treatment.
In a study in 2017, the CDC concluded there were 1.7 million new cases of cancer in the United States. Cancer has surpassed heart disease as the leading cause of death in nearly half of the United States. Gemcitabine is an intravenous antimetabolite used to treat a multitude of cancers. An active inhibitor of ribonucleotide reductase, gemcitabine perturbs natural deoxycytidine formation (2).Although it has a wide range of anticancer activity against solid tumors and a generally manageable toxicity. gemcitabine still presents numerous cytotoxic side effects (3). One current theory apropos the unknown mechanism of gemcitabine’s cytotoxic effect is it being concomitant to the oxidative stress induced by the production of reactive oxygen species. As an antimetabolite, or more specifically, a nucleoside analogue, gemcitabine’s cytotoxicity is correlated with its incorporation into and inhibition of DNA synthesis (4).” This is because gemcitabine is a cytosine derivative, so DNA incorporating gemcitabine is made more susceptible to breakage and replication termination.
Studies have found NAC to be effective in preventing oxidative stress, which is the imbalance of radicals’ production to the body’s ability to counterset their harmful effects (6).Furthermore, NAC enhances gemcitabine efficacy in initially decreasing pancreatic tumor growth, although resistance later develops (7). NAC is a direct thiol antioxidant and replenishes glutathione (GSH), the most prevalent natural antioxidant in the body, to regulate cell signaling under oxidative stress (9). Studies have shown NAC prevents oxidative-stress-caused cell apoptosis by activating signal regulating pathways and interacting with reactive oxygen and nitrogen species as a free-radical scavenger (9).NAC also corrects reduced glutathione (NAC is glutathione’s precursor) concentration, therefore preserving membrane fluidity, and thus relieving oxidative stress.
Prior research demonstrates NAC’s indirect antioxidant effect as a glutathione precursor through reduction of oxidative stress and inflammatory response in COPD patients and the benefits of NAC for administering in HINI influenza pneumonia patients (6, 8). However, little research has been done to investigate the efficacy of NAC in inhibiting cytotoxicity induced by gemcitabine. Given the potent effects of gemcitabine cytotoxicity on healthy cells, can NAC in synergy with gemcitabine abate oxidative stress on cellular membranes and subsequent cytotoxicity? Since gemcitabine activation presents detriments to healthy cells due to stressed cell conditions and NAC counteracts cytotoxicity, it is hypothesized NAC alongside gemcitabine will decrease cell cytotoxicity, a consequence of gemcitabine oxidative stress on cell membranes, and prevent cell death. The addition of the antioxidant, NAC, to gemcitabine-treated yeast cells can determine if oxidative stress is a by-product of gemcitabine and if NAC truly prevents gemcitabine-induced cell death (10). This project can combat gemcitabine-induced cytotoxicity in cells using NAC, hence assisting cancer patients in minimizing healthy cell damage, yielding faster recovery and less chemotherapy side detriments.
MATERIALS AND METHODS
Two separate experiments, spot plating and liquid/turbidity test, were performed. Spot plating was the visual representation of the influence of NAC on gemcitabine on yeast cell growth. Liquid media tests were the statistical representation of the experiment over an eight-hour period. Both experiments utilized Yeast-Extract Peptone Dextrose media and the same concentration of gemcitabine, but varying concentrations of NAC to study its effects on a constant gemcitabine drug level.
Yeast-Extract Peptone Dextrose medium (YEPD) was purchased from Carolina Biological Supply Company. YEPD medium for spot test was prepared by adding 12.5 grams of YEPD. 5 grams of agar, and 250 mL of double distilled water in a sterile flask. YEPD medium for liquid test was prepared by adding 125 grams of YEPD and 250 mL of double distilled water in a sterile fack. Both media were boiled and placed in an incubating water bath for 20 minutes, they were later cooled down.
Prepare Yeast Serial Dilution
Wild type yeast (Saccharomyces cerevisiae), specifically the HAO Strain, was purchased from Carolina Biological Supply Company. Yeast culture was prepared by swabbing yeast from the vial once and adding this sample to 10 mL double distilled water. The culture was mixed well. Then, serial dilutions (concentrations: 1, 0.1, 0.01, 0.001 times original yeast concentration were prepared from the yeast culture and placed in the refrigerator. The same set of serial dilutions was used for the entire experiment.
Spot Plating Test
Samples of gemcitabine (10 mg) and liquid N-acetylcysteine antioxidant (50 mg/mL) were purchased from Millipore Sigma. In labelled falcon tubes, six different concentrations of gemcitabine and NAC were prepared as specified in Table 1. The mouth of the flask containing YEPD media was sterilised using the Bunsen burner flame. Then, 25 mL of YEPD media was added to each of the labeled falcon tubes and invested to mix. The contents of the falcon tubes were poured to the correspondingly labelled petri dishes. Any bubbles that appeared on the surface of the media were removed by quickly moving the Bunsen burner flame over the surface. After the petri dishes were set, 10 µL of each of the yeast culture serial dilutions (1, 0.1, 0.01, 0.001) was pipetted in two rows onto each dish, so that each column consisted of n=2 trials of each dilution setting. Petri dishes were placed in 37 °C to grow overnight. Images of the growth were taken using a digital camera and growth data was recorded after one day
|Petri Dish #||Gemcitabine 10 mg/mL ( µL )||NAC 50 mg/mL ( µL )|
|2 (Gemcitabine control)||100||0|
|6 (NAC control)||0||800|
Liquid Media Test
In labelled falcon tubes, six different concentrations of gemcitabine and NAC were prepared according to Table 2. The mouth of the flask containing YEPD media was sterilized using a Bunsen burner. Then, 15 mL YEPD media and 50 L yeast culture of the first dilution (1 times the original yeast concentration) were added to each of the labeled falcon tubes. The falcon tubes were placed in the agitator. Then, 3.5 mL of each culture was pipetted into its own cuvette to be measured. Every two hours, the optical density at 600nm (OD600) of the culture was measured using a spectrophotometer and recorded for a period of eight hours. After each test, cuvette contents were discarded and fresh media from falcon tubes were used for the new test. Liquid media test was then repeated for the remainder of the yeast culture serial dilutions (0.1, 0.01, 0.001).
|Falcon Tube #||Falcon Gemcitabine 10 mg/mL ( µL )||NAC 50 mg/mL ( µL )|
|2 (Gemcitabine control)||60||0|
|6 (NAC control)||0||120|
Figure 1. Compares the spot plating images of test samples over a growth period of 1 day. For each dish, serial dilutions are organized by column, with n=2 for each specific dilution condition. Dish 1 (control) does not contain gemcitabine or NAC. Dish 2 is treated with 100 µL gemcitabine only to control for gemcitabine. Dish 3, 4, and 5 are treated with 100 µL gemcitabine and varying quantities of NAC (Table 1). Dish 6 contains 800 µL NAC only to control for NAC. Note the lack of growth in Dish 2 and increasing lack of growth in Dishes 3, 4, and 5 in comparison to Dishes 1 and 6.
The spot plating image showed the growth of the yeast cells in different conditions over a time period of one day. The visible amount of cell growth in Dish 2 (gemcitabine 100 µL) was less than the control Dish 1, indicating cell damage due to gemcitabine presence. In Dish 2, serial dilution 0.1 is faintly visible; however, dilutions 0.01 and 0.001 are not visible at all. Thus, compared to the control, Dish 1, which experienced visible growth from all dilutions, it is evident that gemcitabine killed yeast cells (Dish 2). Increasing NAC concentrations allowed more cells to survive. There was more visible cell growth in Dish 3 (gemcitabine 100 µL and NAC 200 µL) through Dish 5 (gemcitabine 100 µL and NAC 800 µL) than Dish 2 (gemcitabine 100 µL). Serial dilutions 1, 0.1, and 0.01 are clearly visible in Dish 5, indicating the prevention of cell death due to NAC presence. Dish 5 was most effective against gemcitabine-induced cell deaths. At a concentration of 800 µL, NAC was most effective in rescuing yeast cells in spot plating with a 100 µL concentration of gemcitabine. Cell growth in Dish 6 that contained only NAC 800 µL was comparable to the control dish, Dish 1.
The graphs of the liquid media results showed the growth trend (OD600) of the yeast cells over a period of eight hours. For each serial dilution, the rate of cell growth in Tube 2 (gemcitabine 60 µL) was consistently less than control Tube 1. On average, 73% of gemcitabine-treated cells in Tube 2 (0.308) were damaged in comparison to the control, Tube 1 (1.106) after eight hours. After adding only 60 µL NAC to the sample, the average cell damage was reduced to 35% in Tube 3 (0.721). With the addition of 90 µL NAC to the sample, the average cell damage was further reduced to 29% in Tube 4 (0.785). NAC was most effective at the highest concentration of 120 µL, which further reduced the cell damage to 18% in Tube 5 (0.900). Tube 6 contained only 120 µL of NAC with no gemcitabine; cell growth (1.058) was 96% and was comparable to the control Tube 1, reaffirming that NAC does not negatively affect cell growth.
Graph 1. Compares the growth (OD600) of NAC and gemcitabine-treated yeast culture (serial dilution 1) samples in liquid media over a time period of eight hours.
Graph 2. Compares the growth (OD600) of NAC and gemcitabine-treated yeast culture (serial dilution 0.1) samples in liquid media over a time period of eight hours.
Graph 3. Compares the growth (OD600) of NAC and gemcitabine-treated yeast culture (serial dilution 0.01) samples in liquid media over a time period of eight hours.
Graph 4. Compares the growth (OD600) of NAC and gemcitabine-treated yeast culture (serial dilution 0.001) samples in liquid media over a time period of eight hours.
|Falcon Tube # and Test Condition||2 Hours||4 Hours||6 Hours||8 Hours|
|2. Gemcitabine 60||0.122||0.132||0.188||0.308|
|3. Gemcitabine 60 & NAC 60||0.130||0.205||0.416||0.721|
|4. Gemcitabine 60 & NAC 90||0.133||0.207||0.501||0.785|
|5. Gemcitabine 60 & NAC 120||0.140||0.224||0.615||0.900|
|6. NAC 120||0.144||0.212||0.770||1.058|
Graph 5. Compares the average growth (OD600) of NAC and gemcitabine-treated yeast culture samples in liquid media from all dilutions over a time period of eight hours. Note that these trends reaffirm the results found with the petri dishes.
Overall, the data from the experiment proved the hypothesis to be accurate: gemcitabine cytotoxicity was due to oxidative stress on cell membranes, and the addition of NAC did prevent apoptosis to a degree. During spot plating, NAC was most effective in preventing yeast cell death at a concentration of 800 µL against a 100 µL concentration of gemcitabine, however, this was also the highest concentration of NAC tested in this study. In the liquid media tests, 120 µL NAC was most effective in preventing apoptosis against 60 µL concentration gemcitabine, however, this was also the highest concentration of NAC in this study. Higher concentration ratios of NAC to gemcitabine could be explored in further studies, as they are likely to have an even stronger protective effect against gemcitabine cytotoxicity. Since there is no precursor to the cytotoxic nature of this experiment, in order to draw stronger conclusions, increasing the sample size and adding more trials for each set of conditions would minimize the variability of data and experimental errors by providing additional data points that enable accurate results and stronger conclusions.
Currently, gemcitabine kills only tumor cells when administered in smaller amounts. However, as the concentrations increase, the drug destroys the tumor cells along with the healthy cells. Therefore, this project aspires to keep drug concentrations low, while optimizing gemcitabine’s cancer-targeting abilities. Future experimentation may include heightening the specificity of NAC-employed cells (so that NAC is only introduced to healthy cells with gemcitabine-induced cytotoxicity, rather than cancer cells, as this would reverse chemotherapy), utilizing different chemotherapy drugs with this experimental method (such as cisplatin) instead of gemcitabine and using other antioxidants, such as vitamin E instead of NAC. Further research can be applied to develop methods of treatment utilizing gemcitabine and NAC in synergy to produce almost negligible side effects.
I would like to thank Mr. Robert Helm, my science project sponsor and advisor, and Mr. Jeremy Walton, my AP Biology teacher, at Methacton High School for help and guidance throughout this project, for reviewing and commenting on the contents of this study, and providing lab facilities.