Metformin is a widely used antidiabetic drug whose anticancer effects, mediated by the activation of AMP-activated protein kinase (AMPK) and reduction of mTOR signaling, have become noteworthy. ...Chemotherapy produces genotoxic stress and induces p53 activity, which can cross-talk with AMPK/mTOR pathway. Herein, we investigate whether the combination of metformin and paclitaxel has an effect in cancer cell lines.
Human tumors were xenografted into severe combined immunodeficient (SCID) mice and the cancer cell lines were treated with only paclitaxel or only metformin, or a combination of both drugs. Western blotting, flow cytometry, and immunohistochemistry were then used to characterize the effects of the different treatments.
The results presented herein show that the addition of metformin to paclitaxel leads to quantitative potentialization of molecular signaling through AMPK and a subsequent potent inhibition of the mTOR signaling pathway. Treatment with metformin and paclitaxel resulted in an increase in the number of cells arrested in the G(2)-M phase of the cell cycle, and decreased the tumor growth and increased apoptosis in tumor-bearing mice, when compared with individual drug treatments.
We have provided evidence for a convergence of metformin and paclitaxel induced signaling at the level of AMPK. This mechanism shows how different drugs may cooperate to augment antigrowth signals, and suggests that target activation of AMPK by metformin may be a compelling ally in cancer treatment.
The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human ...cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.
Genomic instability drives tumorigenesis and DNA repair defects are associated with elevated cancer. Metabolic alterations are also observed during tumorigenesis, although a causal relationship ...between these has not been clearly established. Xeroderma pigmentosum (XP) is a DNA repair disease characterized by early cancer. Cells with reduced expression of the XPC protein display a metabolic shift from OXPHOS to glycolysis, which was linked to accumulation of nuclear DNA damage and oxidants generation via NOX-1. Using XP-C cells, we show that mitochondrial respiratory complex I (CI) is impaired in the absence of XPC, while complex II (CII) is upregulated in XP-C cells. The CI/CII metabolic shift was dependent on XPC, as XPC complementation reverted the phenotype. We demonstrate that mitochondria are the primary source of H
O
and glutathione peroxidase activity is compromised. Moreover, mtDNA is irreversibly damaged and accumulates deletions. XP-C cells were more sensitive to the mitochondrial inhibitor antimycin A, an effect also prevented in XPC-corrected cells. Our results show that XPC deficiency leads to alterations in mitochondrial redox balance with a CI/CII shift as a possible adaptation to lower CI activity, but at the cost of sensitizing XP-C cells to mitochondrial oxidative stress.
Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed ...in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.
BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL ...induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca2+ levels, mitochondrial Ca2+ transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca2+ transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca2+-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca2+ homeostasis and Ca2+-induced opening of MPT pore.
•BlL, a galactose-binding C-type lectin, causes death in cultured B16-F10 melanoma cells.•BlL-induced melanoma cell death involves dysregulation of cellular Ca2+ homeostasis.•BlL-induced melanoma cell death involves opening of mitochondrial permeability transition pore.
The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H
2
O
2
production by isolated rat brain mitochondria in different respiratory ...states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O
2
mg
−1
min
−1
and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O
2
mg
−1
min
−1
in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H
2
O
2
production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H
2
O
2
mg
−1
min
−1
and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H
2
O
2
mg
−1
min
−1
in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP
+
) was used as a respiratory complex I inhibitor. Rotenone-stimulated H
2
O
2
production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H
2
O
2
production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H
2
O
2
production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H
2
O
2
. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.
The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H sub(2)O sub(2) production by isolated rat brain mitochondria in different ...respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 plus or minus 1.0 to 26.4 plus or minus 2.6 nmol O sub(2) mg super(-1) min super(-1) and from 7.8 plus or minus 0.3 to 6.3 plus or minus 0.3 nmol O sub(2) mg super(-1) min super(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H sub(2)O sub(2) production was stimulated from 12.2 plus or minus 1.1 to 21.0 plus or minus 1.2 pmol H sub(2)O sub(2) mg super(-1) min super(-1) and 56.5 plus or minus 4.7 to 95.0 plus or minus 11.1 pmol H sub(2)O sub(2) mg super(-1) min super(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP super(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H sub(2)O sub(2) production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H sub(2)O sub(2) production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H sub(2)O sub(2) production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H sub(2)O sub(2). The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.
The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. ...Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.
Therapeutic angiogenesis would be clinically valuable in situations such as peripheral vascular disease in diabetic patients and tissue reperfusion following ischemia or injury, but approaches using ...traditional isoforms of vascular endothelial growth factor‐A (VEGF) have had little success. The isoform VEGF165 is both soluble and matrix‐associated, but can cause pathologic vascular changes. Freely diffusible VEGF121 is not associated with pathologic angiogenesis, but its failure to remain in the vicinity of the targeted area presents therapeutic challenges. In this study, we evaluate the cellular effects of engineered VEGF variants that tether extracellular VEGF121 to the cell membrane with the goal of activating VEGF receptor 2 (VEGFR2) in a sustained, autologous fashion in endothelial cells. When expressed by primary human retinal endothelial cells (hRECs), the engineered, membrane‐tethered variants eVEGF‐38 and eVEGF‐53 provide a lasting VEGF signal that induces cell proliferation and survival, increases endothelial permeability, promotes the formation of a cord/tube network, and stimulates the formation of elongated filopodia on the endothelial cells. The engineered VEGF variants activate VEGFR2, MAPK/ERK, and the Rho GTPase mediators CDC42 and ROCK, activities that are required for the formation of the elongated filopodia. The sustained, pro‐angiogenic activities induced by eVEGF‐38 and eVEGF‐53 support the potential of engineered VEGF variants‐overexpressing endothelial cells as a novel combination of gene and cell‐based therapeutic strategy for stimulating endothelial cell‐autologous therapeutic angiogenesis.
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