The role of mitochondrial electron transport in tumorigenesis and metastasis.

BACKGROUND: Tumor formation and spread via the circulatory and lymphatic drainage systems is associated with metabolic reprogramming that often includes increased glycolytic metabolism relative to mitochondrial energy production. However, cells within a tumor are not identical due to genetic change, clonal evolution and layers of epigenetic reprogramming. In addition, cell hierarchy impinges on metabolic status while tumor cell phenotype and metabolic status will be influenced by the local microenvironment including stromal cells, developing blood and lymphatic vessels and innate and adaptive immune cells. Mitochondrial mutations and changes in mitochondrial electron transport contribute to metabolic remodeling in cancer in ways that are poorly understood. SCOPE OF REVIEW: This review concerns the role of mitochondria, mitochondrial mutations and mitochondrial electron transport function in tumorigenesis and metastasis. MAJOR CONCLUSIONS: It is concluded that mitochondrial electron transport is required for tumor initiation, growth and metastasis. Nevertheless, defects in mitochondrial electron transport that compromise mitochondrial energy metabolism can contribute to tumor formation and spread. These apparently contradictory phenomena can be reconciled by cells in individual tumors in a particular environment adapting dynamically to optimally balance mitochondrial genome changes and bioenergetic status. GENERAL SIGNIFICANCE: Tumors are complex evolving biological systems characterized by genetic and adaptive epigenetic changes. Understanding the complexity of these changes in terms of bioenergetics and metabolic changes will permit the development of better combination anticancer therapies. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Mitochondrial genome-knockout cells demonstrate a dual mechanism of action for the electron transport complex I inhibitor mycothiazole.

Mycothiazole, a polyketide metabolite isolated from the marine sponge Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC(50) 0.36-13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC(50) 12.2-26.5 µM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ(0) cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G(2)/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

Cell surface oxygen consumption by mitochondrial gene knockout cells.

Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.

Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA.

We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells.

Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ(1)) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ(1), whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40-60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.

Evidence for NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling via plasma membrane electron transport: A sensitive cellular assay for NQO1.

2,3-Dimethoxy 1,4-naphthoquinone (DMNQ), which redox cycles via two-electron reduction, mediates reduction of the cell-impermeative tetrazolium dye WST-1 in kidney epithelial cells (MDCK), which express high levels of NQO1, but not in HL60 or CHO cells, which are NQO1 deficient. DMNQ-dependent WST-1 reduction by MDCK cells was strongly inhibited by low concentrations of the NQO1 inhibitor dicoumarol and was also inhibited by diphenyleneiodonium, capsaicin, and superoxide dismutase (SOD), but not by the uncoupler FCCP or the complex IV inhibitor cyanide. This suggests that DMNQ-dependent WST-1 reduction by MDCK cells is catalyzed by NQO1 via redox cycling and plasma membrane electron transport (PMET). Interestingly, we observed an association between DMNQ/WST-1 reduction and extracellular H(2)O(2) production as determined by Amplex red. Exposure of MDCK cells to DMNQ for 48 h caused cellular toxicity that was extensively reversed by co-incubation with dicoumarol or exogenous SOD, catalase, or N-acetylcysteine. No effects were observed in NQO1-deficient CHO and HL60 cells. In conclusion, we have developed a simple real-time cellular assay for NQO1 and show that PMET plays a significant role in DMNQ redox cycling via NQO1, leading to cellular toxicity in cells with high NQO1 levels.

Effects of mitochondrial gene deletion on tumorigenicity of metastatic melanoma: reassessing the Warburg effect.

Metabolic flexibility is a hallmark of cancer. Although many tumors preferentially use glycolysis in the presence of oxygen for bioenergetic purposes (Warburg effect), the effects of glycolytic metabolism on tumor metastasis have not been investigated. We have employed an extreme model of glycolytic metabolism to investigate the ability of metastatic B16 mouse melanoma cells to grow as primary subcutaneous tumors and to form lung tumors when injected intravenously into syngeneic and immunocompromised mice. Mitochondrial gene-knockout B16rho degrees cells showed delayed subcutaneous tumor growth and, surprisingly, failed to form lung tumors. The results suggest that mitochondrial reactive oxygen species (ROS) may be required for tumor metastasis.

Metabolic flexibility and cell hierarchy in metastatic cancer.

Cancer is characterized by disturbed homeostasis of self-renewing cell populations, and their ability to seed and grow in multiple microenvironments. This overarching cellular property of metastatic cancer emerges from the contentious cancer stem cell hypothesis that underpins the more generic hallmarks of cancer (Hanahan and Weinberg, 2000) and its subsequent add-ons. An additional characteristic, metabolic flexibility, is related to concepts developed by Warburg and to subsequent work by mid 20th century biochemists who elucidated the bioenergetic workings of mitochondria. Metabolic flexibility may circumvent limitations inherent in the increasingly popular but erroneous view that aerobic glycolysis is a universal property of cancer cells. Cancer research in the second half of the 20th century was largely the domain of geneticists and molecular biologists using reductionist approaches. Integrated approaches that address cancer cell hierarchy and complexity, and how cancer cells adapt their metabolism according to their changing environment are now beginning to emerge, and these approaches promise to address the poor mortality statistics of metastatic cancer.

Differential effects of redox-cycling and arylating quinones on trans-plasma membrane electron transport.

Cytotoxicity of quinones has been attributed to free radical generation and to arylation of cellular nucleophiles. For redox-cycling quinones, cell injury is associated with mitochondrial permeability transition, whereas arylating quinones directly depolarise the mitochondrial membrane and deplete ATP. Like mitochondrial electron transport, plasma membrane electron transport (PMET), plays a multifaceted role in cellular redox homeostasis but the effects of quinones on PMET are unknown. Here we investigate the effects of redox-cycling 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), arylating 1,4-benzoquinone (BQ) and mixed mechanism 2-methyl-1,4-naphthoquinone (MNQ) on PMET, viability and growth of P815 mouse mastocytoma cells.BQ and MNQ rapidly and extensively inhibited PMET as determined by WST-1 /mPMS reduction (IC50 3.5-5 microM at 30 min) whereas the effects of DMNQ were less pronounced. In contrast, MTT reduction (cytosolic NADH dehydrogenase activity over 30 min) was weakly inhibited by BQ (IC50 20 microM) but not by MNQ or DMNQ and cell viability was unaffected. Inhibition of WST-1/mPMS reduction by BQ and MNQ but not DMNQ was fully reversed by NAC. Treatment with DMNQ, MNQ and to a lesser extent BQ inhibited cell proliferation as determined by MTT reduction at 48 h. The effects of BQ and MNQ were reversed by NAC through covalent bonding to BQ and MNQ, but not DMNQ. These results show that arylating quinones are more potent inhibitors of PMET than pure redox-cycling quinones, but that redox-cycling quinones are more cytotoxic.