One Sentence Summary

One sentence summary

While investigating the role of glutathione production in RAS transformed cancer cells, I have learned that the tricarboxylic acid cycle intermediate oxaloacetate does not rescue glutamine deprived cells from oxidation, and more work remains to be done to determine the role of the glutamate-cystine antiporter in terms of glutathione production.

Abstract

Glutathione is a tripeptide consisting of cysteine, glycine, and glutamate, synthesized by glutamate-cysteine ligase and glutathione synthase. Being present in millimolar concentrations, it is the primary thiol-based redox buffer in all cells. Knowing how glutathione production affects the tumor microenvironment will add to the understanding of how tumor cells abrogate their excessive ROS levels. Given the fact that RAS transformed cancer cells utilize greater levels of the amino acid glutamine, we deprived lung epithelial carcinoma A549 cells of glutamine and supplemented them with oxaloacetate; however, unlike the compound alpha-ketoglutarate, oxaloacetate was not able to abrogate the oxidative effects of glutamine deprivation due to decreased glutathione production. Another element of glutathione production relies on the import of cysteine, the rate limiting component of glutathione synthesis. Although I attempted to inhibit the xc- glutamate-cystine antiporter by utilizing sulfasalazine, the results were inconclusive and more investigation is required.

Essay

Tumor metabolics is a broad, developing field, and it is certainly no small undertaking to tackle the inner workings of a cancer cell’s metabolism. I went into this project with significant prior knowledge regarding the differences between the metabolisms of RAS oncogene transformed cancer cells and “normal” human cells – one of the most notable of these differences is the utilization of the amino acid glutamine, given the fact that it has been verified that cancer cells exhibit a certain glutamine addiction compared to normal cells. Knowing this and using the knowledge collected by my lab group prior to this summer, I wanted to further investigate the role of glutamine in the production of glutathione, the cell’s primary thiol-based redox buffer. By gathering as much information as possible about glutathione production and how this contributes to cell redox homeostasis, there is more likelihood of finding a divergent point between cancerous and normal cells, giving way for potential treatments for cancer patients and maximizing the utility of existing ones, such as chemotherapy.

In order to further investigate the role of the glutamine carbon in glutathione synthesis and the significance of glutathione synthesis in general, I was responsible for the culture of several cell lines, but the two that were the most relevant to my research questions were Chinese hamster ovary (CHO) cells and lung epithelial carcinoma A549 cells, allowing me to utilize a non-cancerous control (CHO) and a RAS transformed cancer cell line (A549). I was able to monitor the effects of my experiments on the intracellular redox environments through a special intracellular probe that is unique to the laboratory I work in. This redox probe is a Grx1-roGFP2 sensor, utilizing a disulfide bond to produce specific levels of fluorescence when it is either oxidized or reduced. This means that based on the location of this protein sensor (either in the cytosol or mitochondria), a clear understanding of the redox environment can be obtained. If the cell is undergoing oxidative stress, the sensor’s disulfide bond will be broken, releasing a specific fluorescence that signals it is oxidized. The same goesfor when the cell is in an overall reduced environment – the sensor’s disulfide bond will be reduced, releasing a different fluorescent wavelength, allowing for differential analysis using specialized computer programs. By using this sensor, I was able to manipulate the cells I was working with based on deprivation or addition of certain metabolites, inhibition of transporters, and things of that nature while being able to gauge how said manipulations affected the cell’s redox environment, if at all.

With all this in mind, I was able to design multiple experiments that allowed me to gain deeper insight into the inner workings and importance of glutathione synthesis in RAS transformed cancer cells. The first set of experiments I performed over the summer involved depriving the cells of glutamine, an amino acid whose carbons play an important role in the eventual synthesis of glutathione, and thereby the maintenance of intracellular redox homeostasis. It is known through work in our lab that the molecule alpha-ketoglutarate is able to abrogate the effects of the oxidative stress caused by glutamine deprivation, most likely through the conversion of alpha-ketoglutarate to glutamate through the enzymeglutamate dehydrogenase, allowing it to eventually become a component of glutathione. With this in mind, I designed an experiment based on the deprivation of glutamine and the supplementation of the tricarboxylic acid cycle intermediate oxaloacetate with the intention of discovering whether or not oxaloacetate plays some role in glutathione synthesis, or if it can be readily interconverted to glutamine. The experiments consisted of CHO and A549 cells with a full media control, a control that was deprived of glutamine for 24 hours, and cells deprived of glutamine but supplemented with 5 mM oxaloacetate. Upon analysis, when the redox levels of the A549 cells deprived of glutamine were compared to those that were deprived of glutamine but supplemented with oxaloacetate, there was very little difference between the two, meaning that they were both very oxidized (about 49%) compared to the full media control group (23%) and indicating that oxaloacetate is not capable of rescuing the cell of the oxidative effects of glutamine deprivation. Therefore, it is evident that oxaloacetate does not play a significant role in glutathione production, further shedding light on the differences between normal vs. cancer cell metabolism and providing insight to potential therapies that capitalize on these differences.

To further examine the role and mechanisms behind glutathione synthesis in RAS transformed cancer cells, I sought to manipulate a component of its synthesis apart from glutamine. In this set of experiments, I focused on the cysteine component of glutathione; specifically, how the xc- glutamate-cystine antiporter contributes to the synthesis of glutathione and, in turn, the maintenance of redox homeostasis. To accomplish this, I did some research and discovered a potent inhibitor of the xc- antiporter: a compound called sulfasalazine. Using sulfasalazine, I blocked the antiporter and measured the redox levels to see how a lack of cysteine would alter glutathione synthesis, given once the transporter is blocked, cysteine could not be imported into the cell. Upon beginning the experiments, my hypothesis was that blocking this transporter would indeed create an increase in reactive oxygen species (ROS) production, thereby an overall higher level of intracellular oxidation. I ran experiments in CHO and A549 cells with a control, a group with sulfasalazine, and another with sulfasalazine and 10 mM n-acetyl cysteine (NAC) to relieve the cells of the potential oxidation. However, after multiple trials and alterations to the concentration of sulfasalazine and the duration of the treatment period, the results remained inconclusive. I believe that I need to increase the treatment period to 48 hours and use a concentration of 1 mM sulfasalazine in order to see any definitive results, and I unfortunately did not have the time to do so this summer. I plan on running such an experiment in the near future and testing my hypothesis that inhibition of the xc- antiporter via sulfasalazine will lead to higher levels of intracellular oxidation based on the deficit of cysteine, a key component of glutathione, which is the primary thiol-based redox buffer in cells.

Without a doubt, there is much more work to be done in order to uncover all of the inner workings of tumor metabolism, especially where glutathione synthesis is concerned. In the coming semester, I plan to continue my experimentation on the xc- glutamate-cystine antiporter and hopefully garner some meaningful data. Once more is learned about the key differences in normal vs. cancer metabolism, especially in the specific area regarding glutamine and glutathione, strides can be made to capitalize on such differences to maximize the efficacy of cancer treatments and therapies through the high ROS levels present in cancer cells. Hopefully, the research that I have conducted this summer with the aid of SURF will add to the ever-growing body of knowledge on the topic and contribute to such promising therapies.