Introduction: Extracellular signal regulated kinase (ERK) plays a pivotal role on cellular physiology chairing several biological mechanisms. The aim of this study was to better understand the mechanisms underlining a hypothetical mutual correlation between autophagy and proliferative processes in melanoma cell lines. Methods: M14 and 793 melanoma cell lines (ATCC; Manassas, VA, USA) were grown in DMEM and RPMI 1640, respectively, supplemented with FBS, L-glutamine and Pen-Strep at 37 C in 5% CO2. In this study, M14 and 793 cells were starved by low serum treatment (0.1% FBS for 4, 8, 12 and 24 h), or by Rapamycin treatment at different concentrations (10, 50, 75 and 100 nM) or Cloroquine (10 nM for 2, 5, 8 and 12 h). We first evaluated cell viability by MTT, and subsequently p-ERK, p-P70S6 kinase and LC3BII modulation were analyzed by Western Blot and/or IP assays. In addition, confocal and fluorescent microscopy were used to assess the LC3BII expression on M14 and 793 melanoma cells, undergoing 24 h of starvation or 2 h Cloroquine treatment. Results: On M14 and 793 cells undergoing starvation, Rapamycin or Cloroquine treatments showed a significant decrease of viability compared to control group. p-ERK western blots showed a significant modulation with strong ERK activation on M14 cells undergoing 12 h of starvation (p = 0.008) and on 793 melanoma cells after 8 h of starvation (p < 0.001) or 2 h cloroquine (p < 0.001). Western blot analysis didn’t show a p-ERK modulation on M14 and 793 cells treated with Rapamycin. Furthermore, adopting as a positive control, M14 and 793 cells treated with EGF showed a significant increase of p-ERK expression comparable to the control group (p < 0.001), in a time-independent manner. Surprisingly enough, we observed a strong p-p70 expression both on M14 cell line undergoing 12 h and 24 h (p < 0.001) of starvation or treatment with 75 nM Rapamycin (p = 0.01) and with Cloroquine at different time course, 2 h (p = 0.02), 5 h (p = 0.01), 8 h (p = 0.003), and on 793 cells undergoing 12 h and 24 h of starvation (p = 0.01) or treatment with Cloroquine at 8 h and 12 h (p = 0.01) comparable to the control group. Regarding autophagic activation, western blot and confocal microscopy showed a slightly increase of LC3BII in M14 cells undergoing starvation or treated with 2 h of Cloroquine. Conversely 793 cells showed a LC3BII strong modulation, more evident on cells undergoing 24 h of starvation. Conclusion: These findings evidenced a correlation between proliferative and autophagic processes. In fact, we evidenced a higher activation for the following mediators: p-ERK on cells treated with 8 h of starvation and 2 h Cloroquine; p-p70 on cells treated with 12 and 24 h of starvation and with 8 and 12 h Cloroquine; LC3BII on cells undergoing to 12 and 24 h of starvation. These findings evidenced a key role of proliferative pathway in controlling autophagic process. References [1] Wei Y, An Z, Zou Z, Sumpter R, Su M, Zang X, Sinha S, Gaestel M, Levine B. The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. Elife. 2015 Feb 18;4. doi: 10.7554/eLife.05289.

Poster Presentations

FLATI, VINCENZO;
2016

Abstract

Introduction: Extracellular signal regulated kinase (ERK) plays a pivotal role on cellular physiology chairing several biological mechanisms. The aim of this study was to better understand the mechanisms underlining a hypothetical mutual correlation between autophagy and proliferative processes in melanoma cell lines. Methods: M14 and 793 melanoma cell lines (ATCC; Manassas, VA, USA) were grown in DMEM and RPMI 1640, respectively, supplemented with FBS, L-glutamine and Pen-Strep at 37 C in 5% CO2. In this study, M14 and 793 cells were starved by low serum treatment (0.1% FBS for 4, 8, 12 and 24 h), or by Rapamycin treatment at different concentrations (10, 50, 75 and 100 nM) or Cloroquine (10 nM for 2, 5, 8 and 12 h). We first evaluated cell viability by MTT, and subsequently p-ERK, p-P70S6 kinase and LC3BII modulation were analyzed by Western Blot and/or IP assays. In addition, confocal and fluorescent microscopy were used to assess the LC3BII expression on M14 and 793 melanoma cells, undergoing 24 h of starvation or 2 h Cloroquine treatment. Results: On M14 and 793 cells undergoing starvation, Rapamycin or Cloroquine treatments showed a significant decrease of viability compared to control group. p-ERK western blots showed a significant modulation with strong ERK activation on M14 cells undergoing 12 h of starvation (p = 0.008) and on 793 melanoma cells after 8 h of starvation (p < 0.001) or 2 h cloroquine (p < 0.001). Western blot analysis didn’t show a p-ERK modulation on M14 and 793 cells treated with Rapamycin. Furthermore, adopting as a positive control, M14 and 793 cells treated with EGF showed a significant increase of p-ERK expression comparable to the control group (p < 0.001), in a time-independent manner. Surprisingly enough, we observed a strong p-p70 expression both on M14 cell line undergoing 12 h and 24 h (p < 0.001) of starvation or treatment with 75 nM Rapamycin (p = 0.01) and with Cloroquine at different time course, 2 h (p = 0.02), 5 h (p = 0.01), 8 h (p = 0.003), and on 793 cells undergoing 12 h and 24 h of starvation (p = 0.01) or treatment with Cloroquine at 8 h and 12 h (p = 0.01) comparable to the control group. Regarding autophagic activation, western blot and confocal microscopy showed a slightly increase of LC3BII in M14 cells undergoing starvation or treated with 2 h of Cloroquine. Conversely 793 cells showed a LC3BII strong modulation, more evident on cells undergoing 24 h of starvation. Conclusion: These findings evidenced a correlation between proliferative and autophagic processes. In fact, we evidenced a higher activation for the following mediators: p-ERK on cells treated with 8 h of starvation and 2 h Cloroquine; p-p70 on cells treated with 12 and 24 h of starvation and with 8 and 12 h Cloroquine; LC3BII on cells undergoing to 12 and 24 h of starvation. These findings evidenced a key role of proliferative pathway in controlling autophagic process. References [1] Wei Y, An Z, Zou Z, Sumpter R, Su M, Zang X, Sinha S, Gaestel M, Levine B. The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. Elife. 2015 Feb 18;4. doi: 10.7554/eLife.05289.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/111729
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