miR-148a通过靶向调节己糖激酶2基因抑制乳腺癌细胞糖酵解和细胞增殖能力

为了探讨miR-148a及己糖激酶2（hexokinase 2,HK2）基因对人乳腺癌细胞糖酵解代谢途径的影响和可能机制,利用实时荧光定量PCR（real-time fluorescent quantitative PCR,qRT-PCR）检测多种乳腺癌细胞系中miR-148a的表达量,从中筛选miR-148a表达量相对较低的乳腺癌细胞系作为研究对象。再通过观察miR-148a表达量的变化对乳腺癌细胞葡萄糖摄取量、乳酸生成量和细胞增殖指标的影响,以探究miR-148a对乳腺癌细胞糖代谢能力的影响。随后,通过TargetScan在线数据库预测miR-148a和HK2基因的靶向关系,再通过双荧光素酶报告实验、Western免疫印迹以及基因回复实验进行验证,以进一步明确miR-148a和HK2在乳腺癌细胞的糖酵解代谢途径中的作用机制。通过qRT-PCR发现miR-148a在多种乳腺癌细胞系表达降低,尤其是在乳腺癌细胞系MDA-MB231中表达量显著降低（P<0.000 1）。过表达miR-148a使MDA-MB231细胞的葡萄糖摄取量、乳酸生成量、细胞增殖指标均显著下降（P<0.01）;而抑制miR-148a表达使MDA-MB231细胞葡萄糖摄取量、乳酸生成量、细胞增殖指标均显著上升（P<0.01）。通过TargetScan在线数据库预测得出,miR-148a与HK2基因3′非编码区（3′-untranslated region,3′-UTR）具有部分结合位点;而双荧光素酶报告实验发现miR-148a与野生型HK2基因的3′-UTR荧光素酶报告载体结合,不与突变型HK2基因的3′-UTR结合。Western免疫印迹检测结果表明,过表达miR-148a使MDA-MB231细胞中HK2蛋白表达量显著下降（P＜0.000 1）,而抑制miR-148a表达则促进HK2蛋白表达量显著上升（P<0.05）。基因回复实验显示,过表达HK2基因使MDA-MB231乳腺癌细胞的葡萄糖摄取量、乳酸生成量、细胞增殖指标显著上升（P＜0.01）;将过表达miR-148a载体与过表达HK2载体共转染MDA-MB231细胞,miR-148a逆转了HK2所致的葡萄糖摄取量增加和乳酸生成量上升,并抑制细胞增殖。因此,研究提示,miR-148a可通过靶向抑制HK2基因表达而抑制乳腺癌细胞MDA-MB231糖酵解代谢和细胞增殖。     

Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS‐30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D ‐[2‐³H‐glucose] as physiological substrate. Kinetic analysis revealed two components at low‐ and high‐glucose concentrations in both tumor cells. At low glucose and 37°C, the V_max was 55 ± 20 and 17.2 ± 6 nmol (min × mg protein)^?1, whereas the K_m was 0.52 ± 0.7 and 9.3 ± 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC₅₀ = 0.44 ± 0.1; K_i = 0.3 ± 0.1 µM) and phloretin (IC₅₀ = 8.7 µM) in AS‐30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS‐30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS‐30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS‐30D carcinoma and HeLa cells. The much lower GLUT V_max and catalytic efficiency (V_max/K_m) values in comparison to those of G6P‐sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target. J. Cell. Physiol. 221: 552–559, 2009. © 2009 Wiley‐Liss, Inc.     

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3-kinase-dependent but p70S6-kinase-independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin-induced glucose uptake by the use of a chemical compound, ML-9, as well as by transfection with a dominant-negative form of Akt (DeltaAkt). Pretreatment with ML-9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML-9 did not affect insulin-stimulated PI3-kinase nor PKCzeta activities. In consequence, ML-9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, DeltaAkt impaired insulin stimulation of glucose uptake and GFP-tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML-9 treatment for 6 h down-regulated insulin-induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co-transfection of brown adipocytes with DeltaAkt precluded the transactivation of GLUT4-CAT promoter by insulin in a similar fashion as a dominant-negative form of PI3-kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.     

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.     

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.     

Mitochondrial hexokinase from differentiated and undifferentiated HT29 colon cancer cells: effect of some metabolites on the bound/soluble equilibrium

1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.     

Phenethyl isothiocyanate stimulates glucose uptake through the Akt pathway in C2C12 myotubes

Phenethyl isothiocyanate (PEITC) is an aromatic isothiocyanate present in cruciferous vegetables. Several studies have shown that isothiocyanates regulate various intracellular signaling pathways, and thereby show anti-inflammatory and detoxifying activities. However, little is known about the effects of PEITC on glucose metabolism. In this study, we examined whether PEITC promotes glucose utilization in mouse skeletal muscle cells, C2C12 myotubes. PEITC induced glucose uptake, glucose transporter 4 (Glut4) translocation to the plasma membrane, and activation of Akt and ERK in C2C12 cells. Inhibition of Akt suppressed PEITC-induced Glut4 translocation and glucose uptake, whereas ERK inhibition did not. Furthermore, PEITC increased phosphorylation of ErbB2 and ErbB3. Treatment with a pan-ErbB inhibitor reduced Akt activation and the subsequent glucose uptake induced by PEITC. These results indicate that PEITC promotes glucose utilization through the ErbB/Akt pathway in C2C12 myotubes. PEITC may therefore serve as a dietary constituent with beneficial effects on the carbohydrate metabolism.

Abbreviations: PEITC: phenethyl isothiocyanate; Glut4: glucose transporter 4; PI3K: phosphatidylinositide 3-kinase; Nrf2: erythroid?2-related factor; ARE: antioxidant response element; HO?1: heme oxygenase?1; NRG: neuregulin     

BF12, a Novel Benzofuran,Exhibits Antitumor Activity by Inhibiting Microtubules and the PI3K/Akt/mTOR Signaling Pathway in Human Cervical Cancer Cells

BF12 [(2E)‐3‐[6‐Methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐1‐benzofuran‐5‐yl]prop‐2‐enoic acid], a novel derivative of combretastatin A‐4 (CA‐4), was previously found to inhibit tumor cell lines, with a particularly strong inhibitory effect on cervical cancer cells. In this study, we investigated the microtubule polymerization effects and apoptosis signaling mechanism of BF12. BF12 showed a potent efficiency against cervical cancer cells, SiHa and HeLa, with IC₅₀ values of 1.10 and 1.06 μm , respectively. The cellular mechanism studies revealed that BF12 induced G2/M phase arrest and apoptosis in SiHa and HeLa cells, which were associated with alterations in the expression of the cell G2/M cycle checkpoint‐related proteins (cyclin B1 and cdc2) and alterations in the levels of apoptosis‐related proteins (P53, caspase‐3, Bcl‐2, and Bax) of these cells, respectively. Western blot analysis showed that BF12 inhibited the PI3 K/Akt/mTOR signaling pathway and induced apoptosis in human cervical cancer cells. BF12 was identified as a tubulin polymerization inhibitor, evidenced by the effective inhibition of tubulin polymerization and heavily disrupted microtubule networks in living SiHa and HeLa cells. By inhibiting the PI3 K/Akt/mTOR signaling pathway and inducing apoptosis in human cervical cancer cells, BF12 shows promise for use as a microtubule inhibitor.     

Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1

Leptomycin B (LMB) is aStreptomycesmetabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeastcrm1mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of thecrm1mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.     

Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance

Akt is a pro‐survival kinase frequently activated in human cancers and is associated with more aggressive tumors that resist therapy. Here, we connect Akt pathway activation to reduced sensitivity to chemotherapy via Akt phosphorylation of Bax at residue S184, one of the pro‐apoptotic Bcl‐2 family proteins required for cells to undergo apoptosis. We show that phosphorylation by Akt converts the pro‐apoptotic protein Bax into an anti‐apoptotic protein. Mechanistically, we show that phosphorylation (i) enables Bax binding to pro‐apoptotic BH3 proteins in solution, and (ii) prevents Bax inserting into mitochondria. Together, these alterations promote resistance to apoptotic stimuli by sequestering pro‐apoptotic activator BH3 proteins. Bax phosphorylation correlates with cellular resistance to BH3 mimetics in primary ovarian cancer cells. Further, analysis of the TCGA database reveals that 98% of cancer patients with increased BAX levels also have an upregulated Akt pathway, compared to 47% of patients with unchanged or decreased BAX levels. These results suggest that in patients, increased phosphorylated anti‐apoptotic Bax promotes resistance of cancer cells to inherent and drug‐induced apoptosis.     

Cytokine Stimulation Promotes Increased Glucose Uptake Via Translocation at the Plasma Membrane of GLUT1 in HEK293 Cells

Interleukin‐3 (IL‐3) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) are two of the best‐characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL‐3 and GM‐CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL‐3 and GM‐CSF are present in several non‐hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL‐3 and GM‐CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL‐3 and GM‐CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3‐kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non‐insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non‐hematopoietic cells. J. Cell. Biochem. 110: 1471–1480, 2010. © 2010 Wiley‐Liss, Inc.     

RecQL4 cytoplasmic localization: Implications in mitochondrial DNA oxidative damage repair

RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4 when mutated leads to premature aging phenotypes in humans. Unlike other human RecQ helicases, RecQL4 is found both in the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown. In this study, two functional nuclear exporting signals (NESs; pNES2 and pNES3) were identified at the C-terminus of RecQL4. Deletion of pNES2 drastically diminished the cytoplasmic localization of RecQL4. Strikingly, addition of ubiquitination tail at the C-terminus of RecQL4 substantially enriched the cytoplasmic fraction of RecQL4 only in the presence of functional pNES2. Immunofluorescence studies revealed that the cytoplasmic RecQL4 was localized in mitochondria. Consistent with its mitochondrial localization, a regulatory role for RecQL4 in the maintenance of mitochondrial DNA (mtDNA) copy number was demonstrated. Elevation of ectopic expression of RecQL4 increased the mtDNA copy number in HEK293 cells while RecQL4 knock down markedly decreased the mtDNA copy number in U2OS cells. Additionally, a substantially increased level of mitochondrial superoxide production, and a markedly decreased repair capacity for oxidative DNA damage were observed in the mitochondria of both RecQL4 deficient human fibroblasts and RecQL4-suppressed cancer cells. These data strongly suggest a regulatory role for RecQL4 in mitochondrial stability and function. Collectively, our study demonstrates that NES-mediated RecQL4 export to the cytoplasm is essential for the maintenance of mitochondrial genome stability.     

Ganoderic acid Mf and S induce mitochondria mediated apoptosis in human cervical carcinoma HeLa cells

In this work, the effects of a pair of positional isomer of ganoderic acids (GAs), namely ganoderic acid Mf (GA-Mf) and ganoderic acid S (GA-S) purified from the fermented mycelia of Ganoderma lucidum, on induction of cell apoptosis and the apoptotic pathway in HeLa cells were investigated. The results demonstrate that both isomers decreased cell population growth on various human carcinoma cell lines by MTT assay, while GA-Mf had better selectivity between normal and cancer cells. The flow cytometry analysis indicated that treatment of HeLa cells with GA-S caused cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Compared with GA-S, GA-Mf had more potent increase in the number of early and late apoptotic cells. Treatment of HeLa cells with each isomer decreased the mitochondria membrane potential and caused the release of cytochrome c from mitochondria into the cytosol. In addition, stimulation of caspase-3 and caspase-9 activity was observed. The Bax/Bcl-2 ratio was also increased in GA-treated HeLa cells. The results demonstrated that both isomers GA-Mf and GA-S induced apoptosis of human HeLa cells through a mitochondria mediated pathway, but they had the different cell cycle arrest specificity. The findings will be helpful to the development of useful cancer chemopreventive compounds from G. lucidum.     

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.     

Hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells

The flavanone hesperetin is known to decrease basal glucose uptake, although the inhibitory mechanism is largely unknown. Here, we used MDA‐MB‐231 breast cancer cells to investigate the molecular pathways affected by hesperetin. The results indicate that the suppression of glucose uptake is caused by the down‐regulation of glucose transporter 1 (GLUT1). Hesperetin was also found to inhibit insulin‐induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4). In addition, the phosphorylation of the insulin receptor‐beta subunit (IR‐beta) and Akt was suppressed. Hesperetin also decreased cellular proliferation, which is likely due to the inhibition of glucose uptake. Cancer cells are highly dependent on glucose and hesperetin may, therefore, have potential application as an anticancer agent. Copyright © 2012 John Wiley & Sons, Ltd.