Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms.

Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.

Hexokinase II binding to mitochondria is necessary for Kupffer cell activation and is potentiated by ethanol exposure.

Ethanol exposure promotes the development of steatohepatitis, which can progress to end stage liver disease. Kupffer cells have been documented to play a key role in the genesis and progression of alcoholic liver disease with ethanol exposure enhancing Kupffer cell activation. In the present study, we identified the binding of hexokinase II to the mitochondria as a requirement for LPS-induced activation of Kupffer cells and its potentiation by ethanol. LPS and ethanol exposure induced a reduction in sirtuin-3 activity. In turn, the decline of sirtuin-3 activity led to the activation of cyclophilin-D, which mediated an increased binding of hexokinase II to the mitochondria. Suppression of cyclophilin-D expression or enforced detachment of hexokinase II from the mitochondria abrogated the LPS- and ethanol-induced stimulation of Kupffer cells, preventing NADPH oxidase and inflammasome activation. Moreover, activation of AMP-activated protein kinase restored sirtuin-3 activity, thereby preventing LPS and ethanol from stimulating the binding of hexokinase II to the mitochondria and precluding NADPH oxidase and inflammasome activation.

Mitoneet mediates TNFα-induced necroptosis promoted by exposure to fructose and ethanol.

Fructose and ethanol are metabolized principally in the liver and are both known to contribute to the development of hepatic steatosis that can progress to hepatic steatohepatitis. The present study indentifies a synergistic interaction between fructose and ethanol in promoting hepatocyte sensitivity to TNFα-induced necroptosis. Concurrent exposure to fructose and ethanol induces the overexpression of the CDGSH iron-sulfur domain-containing protein 1 (CISD1 or mitoneet), which is localized to the outer mitochondrial membrane. The increased expression of mitoneet primes the hepatocyte for TNFα-induced cytotoxicity. Treatment with TNFα induces the translocation of a Stat3-Grim-19 complex to the mitochondria, which binds to mitoneet and promotes the rapid release of its 2Fe-2S cluster, causing an accumulation of mitochondrial iron. The dramatic increase of mitochondrial iron provokes a surge in formation of reactive oxygen species, resulting in mitochondrial injury and cell death. Additionally, mitoneet is constitutively expressed at high levels in L929 fibrosarcoma cells and is required for L929 cells to undergo TNFα-induced necroptosis in the presence of caspase inhibition, indicating the importance of mitoneet to the necroptotic form of cell death.

Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells.

Hexokinase II (HK2), the enzyme that catalyzes the first committed step of glycolysis, is overexpressed in many cancers, as is the central signaling kinase Akt. Akt activity promotes HK2 association with the mitochondria, as well as glucose uptake by cancer cells. In HeLa cervical cancer cells, Akt inhibitor IV (Ai4) increased nuclear HK2 localization, while in MDA-MB-231 breast cancer cells, Ai4 merely induced cytoplasmic redistribution without increased nuclear accumulation. Small interfering RNA (siRNA) directed against Akt confirmed the effect in HeLa cells. Next, we treated the cells with clotrimazole (CTZ), which detaches HK2 from the mitochondria, or leptomycin B (LMB), which promotes HK2 nuclear accumulation, and determined the effect on HK2 subcellular distribution. In both cell lines, CTZ detached HK2 from the mitochondria, without substantially increasing nuclear HK2, while LMB increased nuclear HK2, without redistributing cytoplasmic HK2. Contrary to expectations, Akt inhibition promoted glucose uptake in both cell lines, suggesting that Akt inhibition may increase glucose uptake by detaching HK2 from the mitochondria. In both cell lines, CTZ and LMB increased glucose uptake. However, the results in the HeLa cells showed greater effects: CTZ increased glucose uptake to a similar degree to Ai4, while LMB was far more effective than either. These data suggest that both detachment of HK2 from the mitochondria and increased nuclear HK2 are important for Ai4-induced increased glucose uptake.

Sirtuin-3 modulates Bak- and Bax-dependent apoptosis.

Sirtuin-3 exhibits properties of a tumor suppressor partly emanating from its ability to control the state of mitochondrial metabolism, with depletion of sirt-3 increasing tumor cell survival. In the present study we demonstrate that depletion of sirtuin-3 brings about an anti-apoptotic phenotype via stimulating cyclophilin-D activity, which promotes the binding of hexokinase II to the mitochondria, thereby preventing Bak/Bax dependent mitochondrial injury and cell death. By contrast, increased expression of sirtuin-3 decreases cyclophilin-D activity, resulting in detachment of hexokinase II from the mitochondria and potentiation of Bak- and Bax-induced mitochondrial injury and loss of cell viability.

Sirtuin-4 modulates sensitivity to induction of the mitochondrial permeability transition pore.

The sustained opening of the mitochondrial permeability transition pore (PTP) is a decisive event in the onset of irreversible cell injury. The PTP is modulated by numerous exogenous and endogenous effectors, including mitochondrial membrane potential, ions and metabolites. Mitochondrial sirtuins have recently emerged as pivotal mediators of mitochondrial metabolism. In the present study, we demonstrate that sirt-4 modulates sensitivity to PTP onset induced by calcium and the oxidative cross linking reagent phenylarsine oxide, and PTP dependent cytotoxicity brought about by TNF or doxorubicin. Moreover, the ability of sirt-4 to modulate onset of the PTP is dependent on the expression of glutamate dehydrogenase-1.

GRIM-19-mediated translocation of STAT3 to mitochondria is necessary for TNF-induced necroptosis.

Tumor necrosis factor (TNF) can induce necroptosis, wherein inhibition of caspase activity prevents apoptosis but initiates an alternative programmed necrosis. The activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK-1) is required for necroptosis to proceed, with suppression of RIPK-1 expression or inhibition of RIPK-1 activity with necrostatin-1 preventing TNF-induced necroptosis. Downstream from the TNF receptor, the generation of reactive oxygen species at the mitochondria has been identified as necessary for the execution of necroptosis; with antioxidants and inhibitors of mitochondrial complex I preventing TNF-induced cytotoxicity. However, components of the signaling pathway that lie between activated RIPK-1 and the mitochondria are unknown. In the study reported here we demonstrate that during TNF-induced necroptosis, STAT3 is phosphorylated on serine 727, which is dependent on RIPK-1 expression or activity. The phosphorylation of STAT3 induces interaction with GRIM-19, a subunit of mitochondrial complex I, with a resultant translocation of STAT3 to the mitochondria, where it induces an increase in reactive oxygen species production and cell death.

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress.

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3ß (GSK3ß) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3ß or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3.

Ethanol increases the vulnerability of mitochondria to induction of the mitochondrial permeability transition (MPT). Cyclophilin-D activity enhances the potential for the permeability transition pore (PTP) to open. In the present study, we demonstrate that ethanol and its metabolism sensitize the PTP to opening, in part by increasing the acetylation and activity of cyclophilin-D. This effect of ethanol is mediated by inhibiting the activity of sirtuin-3, an NAD(+) dependent deacetylase that is localized to the mitochondrial matrix. The ethanol-enhanced acetylation of cyclophilin-D also increases the interaction of cyclophilin-D with the adenine nucleotide translocator-1 (ANT-1) and is dependent on ethanol metabolism. Moreover, activation of AMPK, a known positive modulator of sirtuin activity, prevented the ethanol-induced suppression of sirtuin-3 activity and the attendant increase of cyclophilin-D acetylation, activity and association with ANT-1. Additionally, AMPK reactivation of sirtuin-3 prevented the sensitization to the MPT and the enhancement of cell killing by TNF in cells exposed to ethanol.

Nucleocytoplasmic shuttling of hexokinase II in a cancer cell.

In yeast, the hexokinase type II enzyme (HXKII) translocates to the nucleus in the presence of excess glucose, and participates in glucose repression. However, no evidence has suggested a nuclear function for HXKII in mammalian cells. Herein, we present data showing nuclear localization of HXKII in HeLa cells, both by immunocytochemistry and subcellular fractionation. HXKII is extruded from the nucleus, at least in part, by the activity of the exportin 1/CrmA system, as demonstrated by increased nuclear expression and decreased cytoplasmic expression after incubation with leptomycin B, a bacterially-derived exportin inhibitor. Furthermore, cytoplasmic localization of HXKII is dependent on its enzymatic activity, as inhibiting HXKII activity using 2-deoxy-D-glucose (2DG) increased nuclear localization. This effect was more significant in cells incubated in the absence of glucose for 24 h prior to addition of 2DG. Regulated translocation of HXKII to the nucleus of mammalian cells could represent a previously unknown glucose-sensing mechanism.

Sirtuin-3 deacetylation of cyclophilin D induces dissociation of hexokinase II from the mitochondria.

We demonstrate that the transition from a reliance on glycolysis to oxidative phosphorylation in a transformed cell line is dependent on an increase in the levels and activity of sirtuin-3. Sirtuin-3 deacetylates cyclophilin D, diminishing its peptidyl-prolyl cis-trans isomerase activity and inducing its dissociation from the adenine nucleotide translocator. Moreover, the sirtuin-3-induced inactivation of cyclophilin D causes a detachment of hexokinase II from the mitochondria that is necessary for stimulation of oxidative phosphorylation. These results might have important implications for the role of sirtuin-3 in the metabolism of some cancer cells and their susceptibility to mitochondrial injury and cytotoxicity.

Hexokinase II detachment from the mitochondria potentiates cisplatin induced cytotoxicity through a caspase-2 dependent mechanism.

Cancer cells are frequently glycolytic and overexpress hexokinase II (HXK II). In cancer cells, the majority of hexokinase II is localized to the mitochondria through interaction with the voltage dependent anion channel (VDAC). Disruption in the binding of hexokinase II to the mitochondria has been shown to promote mitochondrial injury provoked by pro-apoptotic proteins. The present study demonstrates that cisplatin induces the PIDD (p53 induced protein with a death domain) dependent activation of caspase-2. In turn, caspase-2 cleaves and activates Bid, resulting in the oligomerization of Bak and the release of cytochrome c. Notably, the detachment of hexokinase II from the mitochondria markedly potentiates the onset of caspase-2 induced mitochondrial damage, thus resulting in a synergistic induction of cisplatin induced cytotoxicity.