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Inhibition of PI3K/AKT/mTOR axis disrupts oxidative stress-mediated survival of melanoma cells.

Hambright HG, Meng P, Kumar AP, Ghosh R - Oncotarget (2015)

Bottom Line: Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level.Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione.NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229, USA.

ABSTRACT
Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level. Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione. Our overall objective was to test whether augmenting this high oxidative stress level in melanoma cells would inhibit their dependence on oncogenic PI3K/AKT/mTOR-mediated survival. We report that NexrutineR augmented the constitutively elevated oxidative stress markers in melanoma cells, which was abrogated by N-acetyl cysteine (NAC) pre-treatment. NexrutineR disrupted growth homeostasis by inhibiting proliferation, survival, and colony formation in melanoma cells without affecting melanocyte cell viability. Increased oxidative stress in melanoma cells inhibited PI3K/AKT/mTOR pathway through disruption of mTORC1 formation and phosphorylation of downstream targets p70S6K, 4EBP1 and rpS6. NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism. Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR.

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Basal ROS and oxidative stress markers in melanoma cells and melanocytes(A) Fluorescent micrographs showing total intracellular ROS by carboxy-H2DCFDA, nuclear counterstain by Hoechst, and merged image in melanocytes (HEMn) and melanoma cells (WM793B, 1205Lu, MeWo) at 10X magnification. (B) Evaluation of basal H2O2-specific ROS by Peroxy Orange 1 (PO-1), 20X magnification. (C) Basal protein levels of PGC1α and NRF2 by western blotting. Quantification of band densitometry is shown below, relative to β-actin loading control. (D) Basal level of oxidized intracellular glutathione (GSSG; nmol/mg protein) determined using luminescence-based assay. (E) Intracellular protein carbonylation used as a measure of protein damage, determined by ELISA. (F) Mitochondrial membrane potentials (ΔΨ) were determined using Nernst equation derivative. Data are presented as means of three independent experiments. Statistical analysis was performed using Student's t-test. Significance values; *indicates p ≤ 0.05; and ***indicates p ≤ 0.001.
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Figure 1: Basal ROS and oxidative stress markers in melanoma cells and melanocytes(A) Fluorescent micrographs showing total intracellular ROS by carboxy-H2DCFDA, nuclear counterstain by Hoechst, and merged image in melanocytes (HEMn) and melanoma cells (WM793B, 1205Lu, MeWo) at 10X magnification. (B) Evaluation of basal H2O2-specific ROS by Peroxy Orange 1 (PO-1), 20X magnification. (C) Basal protein levels of PGC1α and NRF2 by western blotting. Quantification of band densitometry is shown below, relative to β-actin loading control. (D) Basal level of oxidized intracellular glutathione (GSSG; nmol/mg protein) determined using luminescence-based assay. (E) Intracellular protein carbonylation used as a measure of protein damage, determined by ELISA. (F) Mitochondrial membrane potentials (ΔΨ) were determined using Nernst equation derivative. Data are presented as means of three independent experiments. Statistical analysis was performed using Student's t-test. Significance values; *indicates p ≤ 0.05; and ***indicates p ≤ 0.001.

Mentions: We evaluated multiple oxidative stress markers to determine differences between primary melanocytes and melanoma cells. To evaluate the basal ROS levels in the HEMn melanocyte cells and human malignant melanoma cells WM793B, 1205Lu, and MeWo; we used two independent fluorescent dyes. We observed higher levels of oxidized carboxydichlorofluorescein, a measure of total ROS in melanoma cells compared with normal melanocytes (Figure 1A). Quantification is shown in Supplementary Figure 1A. Further, Peroxy Orange-1 (PO-1), which detects H2O2-specific ROS, was also higher in melanoma cells compared with HEMn suggesting that part of the total ROS generated is from H2O2 (Figure 1B). We also analyzed the levels of antioxidant proteins including the master regulator, PGC1α and its target NRF2, which constitutes a primary cellular response to chemical and oxidative stress. Protein levels for PGC1α and NRF2 were higher in all 3 melanoma cells compared with HEMn (Figure 1C). Further, we also found that the NRF2 target, NQO1 was significantly elevated in WM793B, and HMOX1 transcript was elevated in 1205Lu and MeWo compared with HEMn (Suppl. Figure 1B). Subsequently, we determined the overall cell redox status. Under balanced cellular redox conditions, reduced glutathione (GSH) makes up approximately 90% of total glutathione and is constantly converted from the oxidized form (GSSG). Therefore, oxidized glutathione levels are indicative of oxidative stress. We assessed oxidized (GSSG) glutathione levels using a luminescence-based assay. Compared with melanocytes, melanoma cell lines had elevated oxidized glutathione (nmol/mg protein), with the WM793B melanoma cells showing the highest GSSG level (Figure 1D). Overall, oxidized glutathione was significantly elevated (4 to 6 fold) in all melanoma cells compared with melanocytes (Figure 1D). Examination of protein carbonylation, a well-established marker of severe oxidative protein damage showed that all melanoma cells had higher endogenous protein carbonyls compared with melanocytes (Figure 1E). Lastly, we evaluated basal mitochondrial membrane potentials, which reflect intracellular redox homeostasis. In healthy, polarized mitochondria, accumulation of potentiometric dye TMRM can be seen, whereas depolarized mitochondria do not retain the dye and leakage of TMRM is diffused in the cytosol. Confocal imaging of mitochondria allows for quantification of the TMRM fluorescent intensity and used in a Nernst equation derivative, which allows for cellular voltage calculation. We found that all melanoma cell lines had significantly lower basal mitochondrial membrane potentials compared with HEMn cells, indicated by a more positive voltage, which is interpreted as more uncoupled mitochondrial membranes (Figure 1F). Collectively, the data presented in Figure 1 indicate that basal oxidative stress is higher in melanoma cells compared with melanocytes, a feature that might enhance their survival. Therefore, we hypothesized that when melanocytes and melanoma cells are challenged with oxidative stress-inducing agent, the former would exhibit an antioxidant response while the latter would not producing opposite outcomes. To test this hypothesis we used NexrutineR as the oxidative stress-inducing agent.


Inhibition of PI3K/AKT/mTOR axis disrupts oxidative stress-mediated survival of melanoma cells.

Hambright HG, Meng P, Kumar AP, Ghosh R - Oncotarget (2015)

Basal ROS and oxidative stress markers in melanoma cells and melanocytes(A) Fluorescent micrographs showing total intracellular ROS by carboxy-H2DCFDA, nuclear counterstain by Hoechst, and merged image in melanocytes (HEMn) and melanoma cells (WM793B, 1205Lu, MeWo) at 10X magnification. (B) Evaluation of basal H2O2-specific ROS by Peroxy Orange 1 (PO-1), 20X magnification. (C) Basal protein levels of PGC1α and NRF2 by western blotting. Quantification of band densitometry is shown below, relative to β-actin loading control. (D) Basal level of oxidized intracellular glutathione (GSSG; nmol/mg protein) determined using luminescence-based assay. (E) Intracellular protein carbonylation used as a measure of protein damage, determined by ELISA. (F) Mitochondrial membrane potentials (ΔΨ) were determined using Nernst equation derivative. Data are presented as means of three independent experiments. Statistical analysis was performed using Student's t-test. Significance values; *indicates p ≤ 0.05; and ***indicates p ≤ 0.001.
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Figure 1: Basal ROS and oxidative stress markers in melanoma cells and melanocytes(A) Fluorescent micrographs showing total intracellular ROS by carboxy-H2DCFDA, nuclear counterstain by Hoechst, and merged image in melanocytes (HEMn) and melanoma cells (WM793B, 1205Lu, MeWo) at 10X magnification. (B) Evaluation of basal H2O2-specific ROS by Peroxy Orange 1 (PO-1), 20X magnification. (C) Basal protein levels of PGC1α and NRF2 by western blotting. Quantification of band densitometry is shown below, relative to β-actin loading control. (D) Basal level of oxidized intracellular glutathione (GSSG; nmol/mg protein) determined using luminescence-based assay. (E) Intracellular protein carbonylation used as a measure of protein damage, determined by ELISA. (F) Mitochondrial membrane potentials (ΔΨ) were determined using Nernst equation derivative. Data are presented as means of three independent experiments. Statistical analysis was performed using Student's t-test. Significance values; *indicates p ≤ 0.05; and ***indicates p ≤ 0.001.
Mentions: We evaluated multiple oxidative stress markers to determine differences between primary melanocytes and melanoma cells. To evaluate the basal ROS levels in the HEMn melanocyte cells and human malignant melanoma cells WM793B, 1205Lu, and MeWo; we used two independent fluorescent dyes. We observed higher levels of oxidized carboxydichlorofluorescein, a measure of total ROS in melanoma cells compared with normal melanocytes (Figure 1A). Quantification is shown in Supplementary Figure 1A. Further, Peroxy Orange-1 (PO-1), which detects H2O2-specific ROS, was also higher in melanoma cells compared with HEMn suggesting that part of the total ROS generated is from H2O2 (Figure 1B). We also analyzed the levels of antioxidant proteins including the master regulator, PGC1α and its target NRF2, which constitutes a primary cellular response to chemical and oxidative stress. Protein levels for PGC1α and NRF2 were higher in all 3 melanoma cells compared with HEMn (Figure 1C). Further, we also found that the NRF2 target, NQO1 was significantly elevated in WM793B, and HMOX1 transcript was elevated in 1205Lu and MeWo compared with HEMn (Suppl. Figure 1B). Subsequently, we determined the overall cell redox status. Under balanced cellular redox conditions, reduced glutathione (GSH) makes up approximately 90% of total glutathione and is constantly converted from the oxidized form (GSSG). Therefore, oxidized glutathione levels are indicative of oxidative stress. We assessed oxidized (GSSG) glutathione levels using a luminescence-based assay. Compared with melanocytes, melanoma cell lines had elevated oxidized glutathione (nmol/mg protein), with the WM793B melanoma cells showing the highest GSSG level (Figure 1D). Overall, oxidized glutathione was significantly elevated (4 to 6 fold) in all melanoma cells compared with melanocytes (Figure 1D). Examination of protein carbonylation, a well-established marker of severe oxidative protein damage showed that all melanoma cells had higher endogenous protein carbonyls compared with melanocytes (Figure 1E). Lastly, we evaluated basal mitochondrial membrane potentials, which reflect intracellular redox homeostasis. In healthy, polarized mitochondria, accumulation of potentiometric dye TMRM can be seen, whereas depolarized mitochondria do not retain the dye and leakage of TMRM is diffused in the cytosol. Confocal imaging of mitochondria allows for quantification of the TMRM fluorescent intensity and used in a Nernst equation derivative, which allows for cellular voltage calculation. We found that all melanoma cell lines had significantly lower basal mitochondrial membrane potentials compared with HEMn cells, indicated by a more positive voltage, which is interpreted as more uncoupled mitochondrial membranes (Figure 1F). Collectively, the data presented in Figure 1 indicate that basal oxidative stress is higher in melanoma cells compared with melanocytes, a feature that might enhance their survival. Therefore, we hypothesized that when melanocytes and melanoma cells are challenged with oxidative stress-inducing agent, the former would exhibit an antioxidant response while the latter would not producing opposite outcomes. To test this hypothesis we used NexrutineR as the oxidative stress-inducing agent.

Bottom Line: Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level.Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione.NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229, USA.

ABSTRACT
Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level. Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione. Our overall objective was to test whether augmenting this high oxidative stress level in melanoma cells would inhibit their dependence on oncogenic PI3K/AKT/mTOR-mediated survival. We report that NexrutineR augmented the constitutively elevated oxidative stress markers in melanoma cells, which was abrogated by N-acetyl cysteine (NAC) pre-treatment. NexrutineR disrupted growth homeostasis by inhibiting proliferation, survival, and colony formation in melanoma cells without affecting melanocyte cell viability. Increased oxidative stress in melanoma cells inhibited PI3K/AKT/mTOR pathway through disruption of mTORC1 formation and phosphorylation of downstream targets p70S6K, 4EBP1 and rpS6. NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism. Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR.

Show MeSH
Related in: MedlinePlus