- Email: [email protected]
ROS-independent inhibition of melanoma tumor growth by luteolin involves BRAF and the MAPK pathway
S70
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
containing GSH metabolizing enzymes whose translation is controlled by the tRNA methyltransferase alkylation repair homolog 8 (Alkbh8). Epitransciptomic marks, in the form of tRNA modifications, can regulate gene expression at the level of translation. Our studies indicate that defective epitranscriptomic regulation and impairment in selenocysteine (SEC) incorporation, resulting from a deficiency in tRNA methytransferase Alkbh8 (Alkbh8-/-), alters glutathione (GSH) metabolism (4) and triggers a gene signature that is highly predictive (Hazard Ratio 2.65, p o 0.0001) of poor ccRCC survival. We have shown that the Alkbh8-/- gene signature drives both senescence and mitochondrial reprogramming to limit damage that results from defects in mitochondrial reactive oxygen species (ROS) detoxification. Alkbh8-/- mouse embryonic fibroblasts (MEFS) increase many hallmarks of senescence, including senescence associated β-galactosidase, heterocromatic foci, the cyclin dependent kinase inhibitor p16Ink4a, mitochondrial elongation as well as the senescence associated secretory phenotype (SASP). In addition, MEFs undergo a metabolic shift that is highlighted by a striking increase in the level of uncoupling protein 2 (UCP2) which enhances oxygen consumption to reduce mitochondrial ROS burden. Our work defines how defects in Alkbh8 and SEC utilization promote senescence and mitochondrial reprogramming and unveils new mechanistic targets for prolonging ccRCC patient survival.
https://doi.org/10.1016/j.freeradbiomed.2018.10.147
144
ROS-independent inhibition of melanoma tumor growth by luteolin involves BRAF and the MAPK pathway Feng Liu-Smith 1,*, Zi Wang 2, Frank Meyskens 1 1 2
University of California, Irvine, USA Central South University, China
Background: Melanoma is a reactive oxygen species (ROS)-driven cancer but developing antioxidants as therapeutic drugs have not been successful. Methods: We screened a number of antioxidants for their capacity of inhibiting melanoma cell growth and identified luteolin and honokiol as the best candidates. A series of molecular studies were performed in order to delineate the mechanism of action for luteolin. Results: Luteolin unexpectedly induced ROS at its IC50 range of concentrations; co-treatment with antioxidants NAC or mito-TEMPO did not rescue cell growth inhibition, although ROS levels were reduced. Luteolin also inhibited PDE4B and AKT kinase pathways, but over-expression of AKT2 or AKT3 did not show a significant effect on reversing the growth inhibition in vitro. Luteolin showed strong inhibition on BRAF kinase at protein and mRNA levels, accompanied by lower levels of phosphorylated MEK1/2 and ERK1/2 kinases. The BRAF mutation status did not show a difference towards luteolin effect. Over-expression of mutant BRAF (BRAFV600E) in A375 or wild-type BRAF in WM3211 cells reversed the luteolin-induced cell growth inhibition. An in silica pharmacophore mapping revealed that BRAF protein is an excellent target for luteolin. Subsequent protein-ligand docking modeling has revealed that luteolin binds to BRAF through Cys531, a potential redox-sensitive residue that is located in the ATP-binding pocket. Further studies revealed that as a single agent luteolin effectively inhibited melanoma tumor growth in vivo. Conclusion: BRAF may be a crucial and direct target for luteolin, which explains the outstanding inhibitory effect of luteolin in multiple cancer types.
https://doi.org/10.1016/j.freeradbiomed.2018.10.148
145
Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human metastatic melanoma development Fabiana Melo 1,*, Diego Gonçalves 2, Ricardo Sousa 1, Jéssica Gonçalves 2, Douglas Silva 1, Marcelo Icimoto 2, Marcela Gimenez 1, Kátia De Angelis 1, Suzana Llesuy 3, Denise Fernandes 4, Francisco Laurindo 4, Miriam Jasiulionis 2 1
Santa Casa de São Paulo School of Medical Sciences, Brazil UNIFESP, Brazil 3 University of Buenos Aires, Argentina 4 University of São Paulo, Brazil 2
Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2–), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2–, for example by the absence of its cofactor tetrahydrobiopterin (BH4). The present work shows that BH4 synthesis is compromised in melanoma cell lines. Analysis through qPCR showed that the expression of the transcripts for proteins involved in the de novo, salvage and recycling BH4 pathways are altered in melanoma cells when compared to melanocytes, resulting in decreased BH4:BH2 ratio. Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2– levels, leading to NOS coupling, which in turn reduced the cell viability, cell proliferation and the ability of melanoma cells to form tumorspheres. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.
https://doi.org/10.1016/j.freeradbiomed.2018.10.149
146
Effects of fusarochromanone on mitochondrial function in squamous cell carcinoma and salivary gland carcinoma Sumitra Miriyala 1, Ezra Bess 1,*, Sarah Corley 2, Utsab Subedi 1 1 2
Louisiana State University Health Sciences Center, Shreveport, USA University of Louisiana at Monroe, USA
It is known that cancer cells undergo aerobic glycolysis and therefore have a decreased oxidative phosphorylation an increase the level of Reactive Oxygen Species (ROS) in what is called as the Warburg Effect. As both of these physiological processes occur in the mitochondria, this organelle shows potential as a target for terminating cancer cell growth. Fusarochromanone (FC-101) is a fungal metabolite that shows anti-cancer activity by inducing apoptosis and inhibiting the growth of cancer cells invitro by the induction of ROS in the cells. In our project, we studied the effect of FC-101 on the mitochondrial bioenergetics of the head and neck
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
containing GSH metabolizing enzymes whose translation is controlled by the tRNA methyltransferase alkylation repair homolog 8 (Alkbh8). Epitransciptomic marks, in the form of tRNA modifications, can regulate gene expression at the level of translation. Our studies indicate that defective epitranscriptomic regulation and impairment in selenocysteine (SEC) incorporation, resulting from a deficiency in tRNA methytransferase Alkbh8 (Alkbh8-/-), alters glutathione (GSH) metabolism (4) and triggers a gene signature that is highly predictive (Hazard Ratio 2.65, p o 0.0001) of poor ccRCC survival. We have shown that the Alkbh8-/- gene signature drives both senescence and mitochondrial reprogramming to limit damage that results from defects in mitochondrial reactive oxygen species (ROS) detoxification. Alkbh8-/- mouse embryonic fibroblasts (MEFS) increase many hallmarks of senescence, including senescence associated β-galactosidase, heterocromatic foci, the cyclin dependent kinase inhibitor p16Ink4a, mitochondrial elongation as well as the senescence associated secretory phenotype (SASP). In addition, MEFs undergo a metabolic shift that is highlighted by a striking increase in the level of uncoupling protein 2 (UCP2) which enhances oxygen consumption to reduce mitochondrial ROS burden. Our work defines how defects in Alkbh8 and SEC utilization promote senescence and mitochondrial reprogramming and unveils new mechanistic targets for prolonging ccRCC patient survival.
https://doi.org/10.1016/j.freeradbiomed.2018.10.147
144
ROS-independent inhibition of melanoma tumor growth by luteolin involves BRAF and the MAPK pathway Feng Liu-Smith 1,*, Zi Wang 2, Frank Meyskens 1 1 2
University of California, Irvine, USA Central South University, China
Background: Melanoma is a reactive oxygen species (ROS)-driven cancer but developing antioxidants as therapeutic drugs have not been successful. Methods: We screened a number of antioxidants for their capacity of inhibiting melanoma cell growth and identified luteolin and honokiol as the best candidates. A series of molecular studies were performed in order to delineate the mechanism of action for luteolin. Results: Luteolin unexpectedly induced ROS at its IC50 range of concentrations; co-treatment with antioxidants NAC or mito-TEMPO did not rescue cell growth inhibition, although ROS levels were reduced. Luteolin also inhibited PDE4B and AKT kinase pathways, but over-expression of AKT2 or AKT3 did not show a significant effect on reversing the growth inhibition in vitro. Luteolin showed strong inhibition on BRAF kinase at protein and mRNA levels, accompanied by lower levels of phosphorylated MEK1/2 and ERK1/2 kinases. The BRAF mutation status did not show a difference towards luteolin effect. Over-expression of mutant BRAF (BRAFV600E) in A375 or wild-type BRAF in WM3211 cells reversed the luteolin-induced cell growth inhibition. An in silica pharmacophore mapping revealed that BRAF protein is an excellent target for luteolin. Subsequent protein-ligand docking modeling has revealed that luteolin binds to BRAF through Cys531, a potential redox-sensitive residue that is located in the ATP-binding pocket. Further studies revealed that as a single agent luteolin effectively inhibited melanoma tumor growth in vivo. Conclusion: BRAF may be a crucial and direct target for luteolin, which explains the outstanding inhibitory effect of luteolin in multiple cancer types.
https://doi.org/10.1016/j.freeradbiomed.2018.10.148
145
Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human metastatic melanoma development Fabiana Melo 1,*, Diego Gonçalves 2, Ricardo Sousa 1, Jéssica Gonçalves 2, Douglas Silva 1, Marcelo Icimoto 2, Marcela Gimenez 1, Kátia De Angelis 1, Suzana Llesuy 3, Denise Fernandes 4, Francisco Laurindo 4, Miriam Jasiulionis 2 1
Santa Casa de São Paulo School of Medical Sciences, Brazil UNIFESP, Brazil 3 University of Buenos Aires, Argentina 4 University of São Paulo, Brazil 2
Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2–), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2–, for example by the absence of its cofactor tetrahydrobiopterin (BH4). The present work shows that BH4 synthesis is compromised in melanoma cell lines. Analysis through qPCR showed that the expression of the transcripts for proteins involved in the de novo, salvage and recycling BH4 pathways are altered in melanoma cells when compared to melanocytes, resulting in decreased BH4:BH2 ratio. Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2– levels, leading to NOS coupling, which in turn reduced the cell viability, cell proliferation and the ability of melanoma cells to form tumorspheres. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.
https://doi.org/10.1016/j.freeradbiomed.2018.10.149
146
Effects of fusarochromanone on mitochondrial function in squamous cell carcinoma and salivary gland carcinoma Sumitra Miriyala 1, Ezra Bess 1,*, Sarah Corley 2, Utsab Subedi 1 1 2
Louisiana State University Health Sciences Center, Shreveport, USA University of Louisiana at Monroe, USA
It is known that cancer cells undergo aerobic glycolysis and therefore have a decreased oxidative phosphorylation an increase the level of Reactive Oxygen Species (ROS) in what is called as the Warburg Effect. As both of these physiological processes occur in the mitochondria, this organelle shows potential as a target for terminating cancer cell growth. Fusarochromanone (FC-101) is a fungal metabolite that shows anti-cancer activity by inducing apoptosis and inhibiting the growth of cancer cells invitro by the induction of ROS in the cells. In our project, we studied the effect of FC-101 on the mitochondrial bioenergetics of the head and neck