Effect of intratumoral injection of carboplatin combined with pluronic P85 or L61 on experimental colorectal carcinoma in rats

Pluronic, a poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene oxide) block copolymer, has been shown to enhance the cytotoxic activity of anticancer drugs in various cell lines. In the current study the effect of Pluronic P85 (P85) and Pluronic L61 (L61) on the intratumoral chemotherapy of an experimental adenocarcinoma in rats was examined. A total of 120 subcutaneous tumors (4 per rat) were inoculated in 30 BDIX rats and were treated weekly for 4 weeks with intratumoral injection of carboplatin (CPt) alone or with either P85 or L61. Tumors were monitored weekly and were excised at the endpoint for histologic evaluation. The effect of Pluronic on levels of intracellular ATP was explored as a possible mechanism of sensitization. Results showed that tumors treated with low-dose CPt (2.8 mg/kg) and P85 or L61 exhibited significant reductions in tumor volume after 28 days relative to Day 0 (112.7% +/- 34.4%, n = 15; 131.3% +/- 55.6%, n = 8) compared with tumors treated with free drug (339.4% +/- 75.0%, n = 16). Control tumors treated with either P85 or L61 alone or with saline showed volume increases of 1079.4% +/- 143.6% (n = 16), 729.4% +/- 202.2% (n = 7), and 1119.2% +/- 6.1% (n = 16), respectively. Treatment with high-dose CPt (20.7 mg/kg) led to a 79.3% +/- 4.2% reduction in tumor volume, and no differences were noted with addition of P85 or L61. In vitro ATP measurements showed that 28.0 mg/kg of P85 significantly reduced levels of intracellular ATP to 44.7% +/- 1.5% of controls, whereas L61 at this concentration depleted ATP levels completely. Results confirm that Pluronic P85 and L61 act as potent sensitizers to carboplatin chemotherapy of the experimental colorectal carcinoma, leading to a significant reduction of tumor growth compared to carboplatin alone. ATP depletion is a possible mechanism for these observed differences.     

Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP.

Pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) was used for in vitro delivery of [gamma-32P]ATP into intact Jurkat cells. Negatively charged ATP molecules are not able to penetrate cell plasma membrane. Hence, exogenous [gamma-32P]ATP added to a cell culture does not participate in phosphorylation of intracellular proteins. The addition to cells of [gamma-32P]ATP solubilized in positively charged (containing dodecylamine) pluronic micelles results in considerable increase of protein phosphorylation. In this case the treatment of intact cells with alkaline phosphatase (resulting in dephosphorylation of external proteins) causes no essential decrease of [32P]-incorporation in cell proteins. That gives an evidence of delivery of solubilized ATP into a cell. Under the experimental conditions used, pluronic micelles neither influence the viability of cells nor permeabilize cell plasma membrane.     

Di-rhamnolipids improve effect of trehalose on both hypothermic preservation and cryopreservation of rat hepatocytes

Trehalose, a disaccharide of glucose, is a highly hydrophilic small molecule (MW?=?342D) and a bioprotectant normally impermeable to the membrane phospholipid bilayer. Di-rhamnolipids, a major component of rhamnolipids, were applied to increase the effect of trehalose in cryopreservation and hypothermic preservation. We found that di-rhamnolipids (10 mg/L) increased the survival of hepatocytes after cryopreservation or hypothermic preservation as indicated by cell viability using trypan blue exclusion and methyl thiazolyl tetrazolium assay. Correspondingly, after hepatocytes were preserved in the presence of di-rhamnolipids, their hepatospecific functions were comparable to those of freshly cultured cells in terms of intracellular glutathione level, albumin secretion, urea production, and metabolic activities of cytochrome P450 isoforms. Measurement of trehalose intracellular concentration showed that its accumulation increased in the presence of di-rhamnolipids (10 mg/L) but was not altered by two other well-known surfactants, Tween-80, and Pluronic 127. Hence, di-rhamnolipids, which are non-toxic, effective, and commercially available, could be a promising protectant by potentiating the function of trehalose against hypothermic or cryopreservation cell damage.     

The Effect of Combined Exposure of 900 MHz Radiofrequency Fields and Doxorubicin in HL-60 Cells

Human promyelocytic leukemia HL-60 cells were pre-exposed to non-ionizing 900 MHz radiofrequency fields (RF) at 12 µW/cm² power density for 1 hour/day for 3 days and then treated with a chemotherapeutic drug, doxorubicin (DOX, 0.125 mg/L). Several end-points related to toxicity, viz., viability, apoptosis, mitochondrial membrane potential (MMP), intracellular free calcium (Ca²⁺) and Ca²⁺-Mg²⁺ -ATPase activity were measured. The results obtained in un-exposed and sham-exposed control cells were compared with those exposed to RF alone, DOX alone and RF+DOX. The results indicated no significant differences between un-exposed, sham-exposed control cells and those exposed to RF alone while treatment with DOX alone showed a significant decrease in viability, increased apoptosis, decreased MMP, increased Ca²⁺ and decreased Ca²⁺-Mg^2+-ATPase activity. When the latter results were compared with cells exposed RF+DOX, the data showed increased cell proliferation, decreased apoptosis, increased MMP, decreased Ca²⁺ and increased Ca²⁺-Mg²⁺-ATPase activity. Thus, RF pre-exposure appear to protect the HL-60 cells from the toxic effects of subsequent treatment with DOX. These observations were similar to our earlier data which suggested that pre-exposure of mice to 900 MHz RF at 120 µW/cm² power density for 1 hours/day for 14 days had a protective effect in hematopoietic tissue damage induced by subsequent gamma-irradiation.     

Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).     

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞,随机分4组,每组设3个复孔。①正常对照组：常规培养,不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理,并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min,其余同NaHS+ATP组。MTT检测各组细胞活力,荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性,Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比,不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力,NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01）,但NaHS达400 μmol/L浓度时,其保护作用未进一步增加。②随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平,而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力,提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。     

Control of passive permeability of chinese hamster ovary cells by external and intracellular ATP

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.     

In vitro anti-proliferative activities of ellagic acid

The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal fibroblast cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-21% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial growth factor (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung fibroblast cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells.     

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.     

mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis

Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca²⁺ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.     

Differentiation-related changes in mitochondrial properties as indicators of stem cell competence

Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6-fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10-fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/10(3) cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line.     

In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-gamma-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.     

Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome‐c release

Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome‐c (cyt‐c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt‐c release in these events. In accordance with single‐cell experiments, our model showed that loss of cyt‐c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ_m from ?142 to ?88 mV, with active caspase‐3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ_m. However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt‐c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed to Mycoplasma pneumoniae cells or membranes

The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10(5) to 10(7) CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 microgram per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures.     

曲古霉素A增强肺癌细胞对放射线敏感性及机制

目的：探讨组蛋白去乙酰化酶抑制剂曲古霉素A（trichostatin A,TSA）增强人非小细胞肺癌（NscLc）A549对γ-射线敏感性作用及机制。方法：以TSA（0．51zM）预处理细胞18h,再以5Gyγ-射线照射细胞,24h后采用MTT法检测细胞存活率,AnnexinV—PI染色检测细胞凋亡,Westernblot法检测胞浆中和线粒体促凋亡蛋白Bax的表达,流式细胞仪检测细胞线粒体膜电位变化。结果-5Gyγ-射线照射可轻度降低细胞存活率,仅有少量细胞发生凋亡,以TSA预处理再以γ-射线处理细胞,细胞存活率显著下降,凋亡细胞明显增多,伴有线粒体膜电位下降,以及Bax蛋白的激活,表现在线粒体Bax表达较单纯照射组显著增高。结论：TSA通过促进Bax蛋白的活化激活线粒体凋亡途径,增强增强A549细胞对γ-射线的敏感性。     

In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.     

Sublethal hyperthermia enhances anticancer activity of doxorubicin in chronically hypoxic HepG2 cells through ROS-dependent mechanism

Thermal ablation in combination with transarterial chemoembolization (TACE) has been reported to exert a more powerful antitumor effect than thermal ablation alone in hepatocellular carcinoma patients. However, the underlying mechanisms remain unclear. The purpose of the present study was to evaluate whether sublethal hyperthermia encountered in the periablation zone during thermal ablation enhances the anticancer activity of doxorubicin in chronically hypoxic (encountered in the tumor area after TACE) liver cancer cells and to explore the underlying mechanisms. In the present study, HepG2 cells precultured under chronic hypoxic conditions (1% oxygen) were treated in a 42°C water bath for 15 or 30 min, followed by incubation with doxorubicin. Assays were then performed to determine intracellular uptake of doxorubicin, cell viability, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and total antioxidant capacity. The results confirmed that sublethal hyperthermia enhanced the intracellular uptake of doxorubicin into hypoxic HepG2 cells. Hyperthermia combined with doxorubicin led to a greater inhibition of cell viability and increased apoptosis in hypoxic HepG2 cells as compared with hyperthermia or doxorubicin alone. In addition, the combination induced apoptosis by increasing ROS and causing disruption of MMP. Pretreatment with the ROS scavenger N-acetyl cysteine significantly inhibited the apoptotic response, suggesting that cell death is ROS-dependent. These findings suggested that sublethal hyperthermia enhances the anticancer activity of doxorubicin in hypoxic HepG2 cells via a ROS-dependent mechanism.     

Oleate prevents palmitate-induced mitochondrial dysfunction,insulin resistance and inflammatory signaling in neuronal cells

Elevated circulating levels of saturated free fatty acids (sFFAs; e.g. palmitate) are known to provoke inflammatory responses and cause insulin resistance in peripheral tissue. By contrast, mono- or poly-unsaturated FFAs are protective against sFFAs. An excess of sFFAs in the brain circulation may also trigger neuroinflammation and insulin resistance, however the underlying signaling changes have not been clarified in neuronal cells. In the present study, we examined the effects of palmitate on mitochondrial function and viability as well as on intracellular insulin and nuclear factor-κB (NF-κB) signaling pathways in Neuro-2a and primary rat cortical neurons. We next tested whether oleate preconditioning has a protective effect against palmitate-induced toxicity. Palmitate induced both mitochondrial dysfunction and insulin resistance while promoting the phosphorylation of mitogen-activated protein kinases and nuclear translocation of NF-κB p65. Oleate pre-exposure and then removal was sufficient to completely block subsequent palmitate-induced intracellular signaling and metabolic derangements. Oleate also prevented ceramide-induced insulin resistance. Moreover, oleate stimulated ATP while decreasing mitochondrial superoxide productions. The latter were associated with increased levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Inhibition of protein kinase A (PKA) attenuated the protective effect of oleate against palmitate, implicating PKA in the mechanism of oleate action. Oleate increased triglyceride and blocked palmitate-induced diacylglycerol accumulations. Oleate preconditioning was superior to docosahexaenoic acid (DHA) or linoleate in the protection of neuronal cells against palmitate- or ceramide-induced cytotoxicity. We conclude that oleate has beneficial properties against sFFA and ceramide models of insulin resistance-associated damage to neuronal cells.     

肾上腺髓质素和降钙素基因相关肽受体在血管平滑肌细胞的脱敏—受体活性修饰蛋白的作用

新近研究发现,肾上腺髓质素（adrenomedullin,ADM)和降钙素基因相关肽（calcitonin gene-related peptide,CGRP)均能与降钙素受体样受体（calcitoni receptor-like receptor,CRLR)结合,其配体特异性由受体活性修饰蛋白（receptor activity-modifying protein RAMP)调控,本工作在离体培养的大鼠胸主动脉血管平滑肌细胞（vsacular smooth muscle cells,VSMCs)上观察ADM和CGRP受体脱敏现象,以探讨CRLR／RAMP假说在心血管组织方面的意义,用无血清培养基（serum-free medium,SFM)和含有10^-8mol/L ADM,CGRP和肾上腺髓素质前体原N－末端20肽（proadrenomedullin N-terminal 20 peptide PAMP）的SFM培养,再用10^-8mol/L ADM或 CGRP和磷酸二酯酶的抑制剂异丙基次黄苷（isobutyryl methyxanthine,IBMX)与VSMCs进行第二次孵育,然后收集细胞,测定VSMCs cAMP含量。10^-8mol/LADM,CGRP和PAMP单独与VSMCs孵育,VSMCs cAMP含量分别较SFM组高191％（P＜0．01）,385％（P＜0．01）和67％（P＜0．05）,预先用10^-8mol/L ADM ak CGRP与VSMCs孵育可降低随后的CGRP刺激VSMCs产生cAMP,分别较单次CGRP育少44％（P＜0．05）和48％（P＜0．01）,预先用100nmol/L蛋白激酶A（PKA）抑制剂H－89处理VSMCs,可完全阻断ADM和CGRP预处理诱导的第二次CGRP刺激的VSMCs cAMP含量减少,表明VSMCs对CGRP的脱敏过程是通过PKA途径实现的,预先用ADM,CGRP处理VSMCs后,用ADM第二次孵育,细胞内cAMP含量与单次ADM孵育无明显改变,PKA抑制H－89与VSMCs孵育,无论对欠ADM刺激或对ADM和CGRP处理的第二次刺激的cAMP生成均无影响,用PAMP处理VSMCs后,ADM和CGRP的第二次刺激的VSMCs cAMP水平无明显改变（P＞0．05）。结果提示,在离体培养的大鼠VSMCs,ADM epc wsg i euk txgtdmj CGRP受体对预先用ADM和CGRP处理后的激动剂的第二次刺激都脱敏,表明ADM和CGRP的脱敏现象不一致。