Location
LSU Health Sciences Center - New Orleans
Event Website
https://www.medschool.lsuhsc.edu/genetics/2024_medical_student_research_poster_symposium.aspx
Presentation Date
5-11-2024 9:30 AM
Description
BACKGROUND: The semi-essential amino acid L-arginine when metabolized by arginase (ARG2) and NOS2 not only plays a crucial role in the synthesis of polyamines promoting tumor growth but also the production of nitric oxide (NO) and L-citrulline which have anti-tumor capabilities. We have previously demonstrated that stimulation of renal cell carcinoma cells (RCC) with IFN lacking NOS2 did not affect tumor-killing compared to cells expressing it. However, a combination of IFN and LPS has shown to overcome tumor resistance to IFN leading to cell killing. Keeping this in mind, we started another phase of the project to try to understand the mechanisms by which tumor resistance is overcome. We hypothesize that Lcitrulline via ASS/ASL enzymes restores L-arginine availability increasing anti-tumor activity via IFN-NOS2 signaling dependent on tumor heterogeneity.
OBJECTIVES: The main objective is to explore IFN resistant mechanisms in different RCC cell lines and screen new therapeutic drugs. We want to improve our understanding of its resistance mechanisms and to increase the efficacy of combination treatments.
METHODS: Four different murine cell lines, which express different ARG2 and NOS2 levels, were used for our experiments. The cells were cultured in RPMI media containing 1040 of Larginine for 24, 48 hours and stimulated with 100U/ml of IFN. Toll Like receptors (TLR-agonists (10 mg) were also added to the cultures. Culture supernatants were collected at different times to test for L-arginine and L-citrulline (HPLC), and nitrites (Greiss assay). Cellular extracts were obtained and tested (30 g) for ARG2, NOS2, ASS, ASL and GAPDH protein expression (Western blot). ARG activity was measured by enzymatic assays. MTT assay was used to quantify cell viability and proliferation.
RESULTS: At base line, the cell lines presented with different growth patterns with R0 having a higher growth rate. The growth rate appears to be independent of ARG activity. CL-19 grew like R0, but its ARG activity was higher. The nitrate production was higher in CL-19 as compared to the others, and parallels with their sensitivity to IFN When stimulated with IFN, results were variable among the three Renca cells lines, with R2 lacking sensitivity to both IFN and LPS but R0 being equally sensitive to the IFN and LPS combination. There is a delay in Western blot and HPLC results because we had to develop a new HPLC protocol that would allow us to separate L-arginine and L-citrulline which only differ by 0.984g/mol in MW.
CONCLUSIONS: Here, we have found that RCC cell lines present with different patterns in growth, nitrite production, ARG activity, and response to stimulation with IFN. These changes could be correlated to the heterogeneity observed in the tumor microenvironment and tumor resistance. The role of L-citrulline, ASS and ASL as contributors of tumor resistance needs to be further investigated.
Recommended Citation
Arreola, Marisol Mosequeda, "Understanding the tumor microenvironment to overcome resistance in kidney cancer" (2024). Medical Student Research Poster Symposium. 5.
https://digitalscholar.lsuhsc.edu/sommrd/2024MSRD/Posters/5
Included in
Understanding the tumor microenvironment to overcome resistance in kidney cancer
LSU Health Sciences Center - New Orleans
BACKGROUND: The semi-essential amino acid L-arginine when metabolized by arginase (ARG2) and NOS2 not only plays a crucial role in the synthesis of polyamines promoting tumor growth but also the production of nitric oxide (NO) and L-citrulline which have anti-tumor capabilities. We have previously demonstrated that stimulation of renal cell carcinoma cells (RCC) with IFN lacking NOS2 did not affect tumor-killing compared to cells expressing it. However, a combination of IFN and LPS has shown to overcome tumor resistance to IFN leading to cell killing. Keeping this in mind, we started another phase of the project to try to understand the mechanisms by which tumor resistance is overcome. We hypothesize that Lcitrulline via ASS/ASL enzymes restores L-arginine availability increasing anti-tumor activity via IFN-NOS2 signaling dependent on tumor heterogeneity.
OBJECTIVES: The main objective is to explore IFN resistant mechanisms in different RCC cell lines and screen new therapeutic drugs. We want to improve our understanding of its resistance mechanisms and to increase the efficacy of combination treatments.
METHODS: Four different murine cell lines, which express different ARG2 and NOS2 levels, were used for our experiments. The cells were cultured in RPMI media containing 1040 of Larginine for 24, 48 hours and stimulated with 100U/ml of IFN. Toll Like receptors (TLR-agonists (10 mg) were also added to the cultures. Culture supernatants were collected at different times to test for L-arginine and L-citrulline (HPLC), and nitrites (Greiss assay). Cellular extracts were obtained and tested (30 g) for ARG2, NOS2, ASS, ASL and GAPDH protein expression (Western blot). ARG activity was measured by enzymatic assays. MTT assay was used to quantify cell viability and proliferation.
RESULTS: At base line, the cell lines presented with different growth patterns with R0 having a higher growth rate. The growth rate appears to be independent of ARG activity. CL-19 grew like R0, but its ARG activity was higher. The nitrate production was higher in CL-19 as compared to the others, and parallels with their sensitivity to IFN When stimulated with IFN, results were variable among the three Renca cells lines, with R2 lacking sensitivity to both IFN and LPS but R0 being equally sensitive to the IFN and LPS combination. There is a delay in Western blot and HPLC results because we had to develop a new HPLC protocol that would allow us to separate L-arginine and L-citrulline which only differ by 0.984g/mol in MW.
CONCLUSIONS: Here, we have found that RCC cell lines present with different patterns in growth, nitrite production, ARG activity, and response to stimulation with IFN. These changes could be correlated to the heterogeneity observed in the tumor microenvironment and tumor resistance. The role of L-citrulline, ASS and ASL as contributors of tumor resistance needs to be further investigated.
https://digitalscholar.lsuhsc.edu/sommrd/2024MSRD/Posters/5
Comments
Award for Cancer Research, LSU LCMC Health Cancer Center Travel Award, Freshman/Sophomore Category
Mentor: Dr. Arnold H. Zea, PhD LSUHSC, Stanley S. Scott Cancer Center; Department of Microbiology