Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

BACKGROUND AND PURPOSE: Many disparate studies have reported the ambiguous role of hydrogen sulfide (H2 S) in cell survival. The present study investigated the effect of H2 S on the viability of cancer and non-cancer cells. EXPERIMENTAL APPROACH: Cancer and non-cancer cells were exposed to H2 S [using sodium hydrosulfide (NaHS) and GYY4137] and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi ) was determined by ratiometric pHi measurement using BCECF staining. KEY RESULTS: Continuous, but not a single, exposure to H2 S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H2 S-releasing donor, GYY4137, significantly increased glycolysis, leading to overproduction of lactate. H2 S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification, leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells. CONCLUSIONS AND IMPLICATIONS: Low and continuous exposure to H2 S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

Altered dynamics of ubiquitin hybrid proteins during tumor cell apoptosis.

The ubiquitin hybrid genes Uba80 and Uba52 encode ubiquitin (Ub), which is fused to the ribosomal proteins S27a (RPS27a) and L40 (RPL40), respectively. Here, we show that these genes are preferentially over-expressed during hepatoma cell apoptosis. Experiments using the tet-inducible transgenic system revealed that over-expression of the ubiquitin hybrid genes sensitized the cells to apoptosis. Further analysis suggested that Ub, and not RPS27a or RPL40, was associated with apoptotic cell death. Cleavage-resistant mutation analysis revealed that the N-terminal portion and the last two amino acids (GG) of Ub are critical for cleavage at the junction between the two protein moieties. An apoptogenic stimulus enhances the nuclear targeting and aggregation of Ub in the nucleus, resulting in histone H2A deubiquitylation followed by abnormal ubiquitylation of the nuclear envelope and the lamina. These events accompany the apoptotic nuclear morphology in the late stage of apoptosis. Each fused RP is localized in the nucleoli. These results suggest a role for Ub hybrid proteins in the altered nuclear dynamics of Ub during tumor cell apoptosis induced by apoptogenic stimuli.

Genotoxicity evaluation of electromagnetic fields generated by 835-MHz mobile phone frequency band.

It is still unclear whether the exposure to electromagnetic fields (EMFs) generated by mobile phone radiation is directly linked to cancer. We examined the biological effects of an EMF at 835 MHz, the most widely used communication frequency band in Korean CDMA mobile phone networks, on bacterial reverse mutation (Ames assay) and DNA stability (in vitro DNA degradation). In the Ames assay, tester strains alone or combined with positive mutagen were applied in an artificial mobile phone frequency EMF generator with continuous waveform at a specific absorption rate (SAR) of 4 W/kg for 48 h. In the presence of the 835-MHz EMF radiation, incubation with positive mutagen 4-nitroquinoline-1-oxide and cumene hydroxide further increased the mutation rate in Escherichia coli WP2 and TA102, respectively, while the contrary results in Salmonella typhimurium TA98 and TA1535 treated with 4-nitroquinoline-1-oxide and sodium azide, respectively, were shown as antimutagenic. However, these mutagenic or co-mutagenic effects of 835-MHz radiation were not significantly repeated in other relevant strains with same mutation type. In the DNA degradation test, the exposure to 835-MHz EMF did not change the rate of degradation observed using plasmid pBluescript SK(+) as an indicator. Thus, we suggest that 835-MHz EMF under the conditions of our study neither affected the reverse mutation frequency nor accelerated DNA degradation in vitro.

The role of tissue transglutaminase in the germinal vesicle breakdown of mouse oocytes.

We have investigated the novel function of tissue transglutaminase (tTG) in the germinal vesicle breakdown (GVBD) of mouse oocyte. tTG was identified in ooplasm and germinal vesicle by immunostaining with less amount in germinal vesicle. Spontaneous maturation of the oocytes elevated in situ activity of tTG by over 2.5-fold at 3 h, which was determined by a confocal microscopic assay. However, incubation with monodansylcadaverine (MDC), a tTG inhibitor, blocked the activation of tTG. The possible role of tTG in GVBD was investigated by the use of two tTG inhibitors, MDC and cystamine. MDC largely inhibited the GVBD by a concentration-dependent manner. GV-stage oocytes were matured to the GVBD stage by 78% at 3 h in the normal culture condition. However, in the oocytes incubated with MDC for 3 h, the GVBD rates were 43 and 11% by 50 and 100 microM, respectively. MDC also blocked the entry of 70 kDa RITC-dextran from the ooplasm to the compartment of germinal vesicle, indicating a possible inhibition of nuclear pore disassembly by MDC. The role of tTG in GVBD was further investigated by microinjection with cystamine. The control oocytes, injected with DPBS, showed about 80% of GVBD at 3 h. But the oocytes injected with cystamine showed 15% of GVBD at 3 h and a little higher rate at 6 h. In addition, the inhibition of GVBD maturation by MDC was reversible by washing. These results suggested that tTG was involved in the early event of mouse oocyte maturation.

Real-time measurement of intracellular reactive oxygen species using Mito tracker orange (CMH2TMRos).

We have investigated a novel method to monitor real changes of intracellular ROS by the use of CMH2TMRos (a reduced form of MitoTracker orange) in Swiss 3T3 fibroblasts. Arachidonic acid induced a rapid increase of CMTMRos fluorescence with a maximal elevation at 120-150 sec, which was determined by scanning every 10 sec with a confocal microscope. The fluorescence increase by arachidonic acid was completely inhibited by 2-MPG but not by catalase, indicating a major contribution of superoxide to the oxidation of CMH2TMRos. Incubation with glucose oxidase, exogenous H2O2, KO2 and lysophosphatidic acid also increased the CMTMRos fluorescence, which was blocked by 2-MPG. These results suggested that CMH2TMRos is a useful fluorophore for real-time monitoring of intracellular ROS and also indicated that CMH2TMRos detects primarily superoxide in cells even though the fluorophore can be oxidized by both superoxide and H2O2.

Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression.

Stimulation of human lung fibroblast cells with TGF-beta1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-l -cysteine (NAC). TGF-beta1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-beta1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-beta1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca2+ influx is stimulated by TGF-beta1 treatment. EGTA suppressed TGF-beta1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-beta1- or H2O2-induced IL-6 and AP-1 activation. In addition, TGF-beta1 or H2O2 increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-beta1-induced IL-6 expression. Taken together, these results indicate that TGF-beta1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, and AP-1 activation and IL-6 gene expression.

Involvement of cytosolic phospholipase A2, and the subsequent release of arachidonic acid, in signalling by rac for the generation of intracellular reactive oxygen species in rat-2 fibroblasts.

Although there have been a number of recent studies on the role of Rac in the generation of reactive oxygen species (ROS), details of the signalling pathway remain unclear. In the present study we analysed the extent to which the activation of cytosolic phospholipase A(2) and the resultant release of arachidonic acid (AA) are involved in the Rac-mediated generation of ROS. Transfection of Rat-2 cells with RacV12, a constitutively active form of Rac1, induced elevated levels of ROS, as reflected by increased H(2)O(2)-sensitive fluorescence of 2', 7'-dichlorofluorescein. These effects could be blocked by inhibiting phospholipase A(2) or 5-lipoxygenase but not by inhibiting cyclo-oxygenase. The application of exogenous AA increased levels of ROS but the effect was dependent on the further metabolism of AA to leukotrienes C(4)/D(4)/E(4) by 5-lipoxygenase. Indeed, the exogenous application of a mixture of leukotrienes C(4)/D(4)/E(4) elicited transient elevations in the levels of ROS that were blocked by catalase. These findings indicate that phospholipase A(2) and subsequent AA metabolism by 5-lipoxygenase act as downstream mediators in a Rac signalling pathway leading to the generation of ROS.

The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts.

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca(2+) ([Ca(2+)](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca(2+)](i), and sustained Ca(2+) increase disappeared in the absence of extracellular Ca(2+). However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca(2+)](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H(2)O(2), with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H(2)O(2) scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca(2+) response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca(2+) response to extracellular H(2)O(2) and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca(2+)](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca(2+) response to EGF, but not vice versa. These results suggested that intracellular H(2)O(2) regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca(2+)](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca(2+)](i) by producing intracellular H(2)O(2).

Hydrogen peroxide activates p70(S6k) signaling pathway.

We investigated a possible role of reactive oxygen species (ROS) in p70(S6k) activation, which plays an important role in the progression of cells from G(0)/G(1) to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H(2)O(2) generated extracellularly by glucose/glucose oxidase led to the activation of p70(S6k) and p90(Rsk) and to phosphorylation of p42(MAPK)/p44(MAPK). The activation of p70(S6k) and p90(Rsk) was dose-dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70(S6k) using specific inhibitors for p70(S6k) signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin-sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70(S6k) activity. In addition, Ca(2+) chelation also inhibited ROS-induced activation of p70(S6k), indicating that Ca(2+) is a mediator of p70(S6k) activation by ROS. However, down-regulation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70(S6k) by ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70(S6k) activity by H(2)O(2) in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H(2)O(2), phosphorylation, and activation of p70(S6k), which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70(S6k) signaling pathway.

Roles of RhoA and phospholipase A2 in the elevation of intracellular H2O2 by transforming growth factor-beta in Swiss 3T3 fibroblasts.

We have investigated the mechanisms by which transforming growth factor-beta (TGF-beta) increased intracellular H2O2 in Swiss 3T3 fibroblasts. Increase of intracellular H2O2 by TGF-beta was maximal at 30 min and blocked by catalase from Aspergillus niger. Scrape-loading of C3 transferase, which down-regulated RhoA, inhibited the production of H2O2 in response to TGF-beta. TGF-beta stimulated release of arachidonic acid, which was completely inhibited by mepacrine, a phospholipase A2 inhibitor. Mepacrine also blocked the increase of H2O2 by TGF-beta. In addition, arachidonic acid increased intracellular H2O2. Furthermore, TGF-beta stimulated stress fibre formation, which was blocked by catalase, without membrane ruffling. Catalase also inhibited stimulation of thymidine incorporation by TGF-beta. These results suggested that TGF-beta increased intracellular H2O2 through RhoA and phospholipase A2, and also suggested that intracellular H2O2 was required for the stimulation of stress fibre formation and DNA synthesis in response to TGF-beta.

Lysophosphatidic acid increases intracellular H2O2 by phospholipase D and RhoA in rat-2 fibroblasts.

We have investigated the possible roles of phospholipase D (PLD) and RhoA in the production of intracellular H2O2 and actin polymerization in response to lysophosphatidic acid (LPA) in Rat-2 fibroblasts. LPA increased intracellular H2O2, with a maximal increase at 30 min, which was blocked by the catalase from Aspergillus niger. The LPA-stimulated production of H2O2 was inhibited by 1-butanol or PKC-downregulation, but not by 2-butanol. Purified phosphatidic acid (PA) also increased intracellular H2O2 and the increase was inhibited by the catalase. The role of RhoA was studied by the scrape-loading of C3 transferase into the cells. The C3 toxin, which inhibited stress fiber formation stimulated by LPA, blocked the H2O2 production in response to LPA or PA, but had no inhibitory effect on the activation of PLD by LPA. Exogenous H2O2 increased F-actin content by stress fiber formation. In addition, catalase inhibited actin polymerization activated by LPA, PA, or H2O2, indicated the role of H2O2 in actin polymerization. These results suggest that LPA increased intracellular H2O2 by the activation of PLD and RhoA, and that intracellular H2O2 was required for the LPA-stimulated stress fiber formation.

Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli.

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

Phosphatidic acid-induced elevation of intracellular Ca2+ is mediated by RhoA and H2O2 in Rat-2 fibroblasts.

We have investigated possible roles of RhoA and H2O2 in the elevation of intracellular Ca2+ ([Ca2+]i) by phosphatidic acid (PA) in Rat-2 fibroblasts. PA induced a transient elevation of [Ca2+]i in the presence or absence of EGTA. Lysophosphatidic acid (LPA) also increased [Ca2+]i, but the sustained Ca2+ response was inhibited by EGTA. LPA stimulated the production of inositol phosphates, but PA did not. In the presence of EGTA, preincubation with LPA completely blocked the subsequent elevation of [Ca2+]i by PA, but not vice versa. PA stimulated the translocation of RhoA to the particulate fraction as did LPA. Scrape loading of C3 transferase inhibited the transient Ca2+ response to PA, but not to LPA, suggesting an essential role of RhoA in the elevation of [Ca2+]i by PA. H2O2 also induced a transient increase of [Ca2+]i as did PA. H2O2 scavengers, catalase and N-acetyl-L-cysteine, completely blocked the rise of [Ca2+]i stimulated by PA, but not by LPA. Furthermore, preincubation with PA blocked the subsequent Ca2+ response to H2O2, and the incubation with H2O2 also blocked the PA-induced rise of [Ca2+]i. Thus, it was suggested that PA stimulated Ca2+ release from PA-sensitive, but not inositol 1,4,5-trisphosphate-sensitive, Ca2+ stores by the activation of RhoA and intracellular H2O2.