Non-metabolic enzyme function of PKM2 in hepatocellular carcinoma: A review.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors with the highest incidence and mortality in the world, causing a serious burden on society. Pyruvate kinase M2 (PKM2) is one of the principal metabolic enzymes involved in glycolysis. Studies have shown that PKM2 is highly expressed in HCC and can be translocated to the nucleus, where it interacts with various transcription factors and proteins such as hypoxia-inducible factor-1α, sterol regulatory element-binding protein 1a, signal transducer and activator of transcription 3, nuclear factor erythroid 2-like 2 and histone H3, exerting non-metabolic enzyme functions to regulate the cell cycle, proliferation, apoptosis, immune escape, migration, and invasion, as well as HCC angiogenesis and tumor microenvironment. This review is focused on the recent progress of PKM2 interacting with various transcription factors and proteins affecting the onset and development of HCC, as well as natural drugs and noncoding RNA impacting diverse biological functions of liver cancer cells by regulating PKM2 non-metabolic enzyme functions, thereby providing valuable directions for the prognosis improvement and molecular targeted therapy of HCC in the future.

Evaluation of the possible association of PDCD-1 and LAG3 gene polymorphisms with hepatocellular carcinoma risk.

PURPOSE: Programmed death-1 (PDCD-1) and lymphocyte activating 3 (LAG3), two important immunosuppressive molecules, play crucial roles in immune escape of tumor cells. This study evaluated the effects of PDCD-1 (rs10204525 and rs36084323), and LAG3 (rs870849 and rs1882545) gene polymorphisms on hepatocellular carcinoma (HCC) risk. METHODS: 341 patients with HCC and 350 cancer-free controls in the South Chinese population were included in a population-based case-control study. DNAs were extracted from peripheral blood samples. Genotypes were analyzed using multiplex PCR and sequencing. SNPs were analyzed using multiple inheritance models (co-dominant, dominant, recessive, and over-dominant). RESULTS: The allele and genotype frequencies of neither of the four polymorphisms, adjusted for age and gender, differed between HCC patients and controls. The differences were also not significant after stratifying by gender and age. According to our results, HCC patients with rs10204525 TC genotype had significantly lower AFP levels than HCC patients with rs10204525 TT genotype (P = 0.004). Moreover, the frequency of PDCD-1 rs36084323 CT genotype reduced the risk of TNM grade (CT vs. C/C-T/T: OR = 0.57, 95%CI = 0.37-0.87, P = 0.049). CONCLUSION: Our results demonstrated that the PDCD-1 (rs10204525 and rs36084323), and LAG3 (rs870849 and rs1882545) polymorphism did not influence the risk of HCC, PDCD-1 rs10204525 TC genotype was associated with the lower AFP levels and rs36084323 CT genotypes were related to HCC tumor grades in the South Chinese samples.

Protective effect of sodium ferulate on acetaldehyde-treated precision-cut rat liver slices.

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis, and inhibition of HSC activation may prevent liver fibrosis. Acetaldehyde, the most deleterious metabolite of alcohol, triggers HSC activation in alcoholic liver injury. In the present study, we investigated the protective effect of sodium ferulate (SF), a sodium salt of ferulic acid that is rich in fruits and vegetables, on acetaldehyde-stimulated HSC activation using precision-cut liver slices (PCLSs). Rat PCLSs were co-incubated with 350 µM acetaldehyde and different concentrations of SF. Hepatotoxicity was assessed by measuring enzyme leakage and malondialdehyde content in tissue. α-Smooth muscle actin, transforming growth factor-ß(1), and hydroxyproline were determined to assess the activation of HSCs. In addition, matrix metalloproteinase (MMP)-1 and the tissue inhibitor of metalloproteinase (TIMP-1) were determined to evaluate collagen degradation. SF prominently prevented the enzyme leakage in acetaldehyde-treated slices and also inhibited HSC activation and collagen production stimulated by acetaldehyde. In addition, SF increased MMP-1 expression and decreased TIMP-1 expression. These results showed that SF protected PCLSs from acetaldehyde-stimulated HSC activation and liver injury, which may be associated with the attenuation of oxidative injury and acceleration of collagen degradation.

Indole-3-carbinol inhibits hepatic stellate cells proliferation by blocking NADPH oxidase/reactive oxygen species/p38 MAPK pathway.

During the course of liver fibrogenesis, hepatic stellate cell (HSC) proliferation as well as subsequent synthesis of excessive extracellular matrix components is known to be the central events. Thus, factors that could limit HSC proliferation are potential anti-fibrotic agents. The aim of this study was to investigate the effects of indole-3-carbinol (I3C), a nutritional component derived from Brassica family vegetables, on the proliferation of cultured HSC and to clarify the underline molecular mechanism. HSC-T6, an activated rat HSC line, was treated with I3C (50, 100 and 200 µM) for 24 h. The results indicated that I3C can significantly inhibit HSC proliferation in a concentration-dependent manner with or without platelet-derived growth factor-BB (PDGF-BB) stimulation (P<0.01). I3C could also block HSC in the G(0)/G(1) phase from entering the S phase. The expressions of α-smooth muscle actin in HSC treated with I3C, were significantly decreased at levels of protein and mRNA (P<0.01). In addition, the type I collagen level, cyclin D(1) and cyclin-dependent kinase 4 mRNA expressions, intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and reactive oxygen species generation were significantly decreased by I3C (P<0.05). We also observed that the phosphorylated p38 MAPK in HSC-T6 was inhibited by I3C in a concentration-dependent manner; however, the phosphorylated ERK1/2 was unaltered. In conclusion, I3C could inhibit the proliferation of HSC by blocking the NADPH oxidase/reactive oxygen species/p38 MAPK signal pathway. These findings suggest that dietary I3C might play a novel role in prevention and treatment of chronic liver diseases.

Effect of indole-3-carbinol on ethanol-induced liver injury and acetaldehyde-stimulated hepatic stellate cells activation using precision-cut rat liver slices.

1. Indole-3-carbinol (I3C), a major indole compound found in high levels in cruciferous vegetables, shows a broad spectrum of biological activities. However, few studies have reported the effect of I3C on alcoholic liver injury. In the present study, we investigated the protective effect of I3C on acute ethanol-induced hepatotoxicity and acetaldehyde-stimulated hepatic stellate cells (HSC) activation using precision-cut liver slices (PCLS). 2. Rat PCLS were incubated with 50 mmol/L ethanol or 350 µmol/L acetaldehyde, and different concentrations (100-400 µmol/L) of I3C were added into the culture system of these two liver injury models, respectively. Hepatotoxicity was assessed by measuring enzyme leakage and malondialdehyde (MDA) content in tissue. Activities of alcoholic enzymes were also determined. α-Smooth muscle actin (α-SMA), transforming growth factor (TGF-ß(1) ) and hydroxyproline (HYP) were used as indices to evaluate the activation of HSC. In addition, matrix metalloproteinase-1 (MMP-1) and the tissue inhibitor of metalloproteinase (TIMP-1) were observed to estimate collagen degradation. 3. I3C significantly reduced the enzyme leakage in ethanol-treated slices. In I3C groups, cytochrome P450 (CYP) 2E1 activities were inhibited by 40.9-51.8%, whereas alcohol dehydrogenase (ADH) activity was enhanced 1.6-fold compared with the ethanol-treated group. I3C also showed an inhibitory effect against HSC activation and collagen production stimulated by acetaldehyde. After being incubated with I3C (400 µmol/L), the expression of MMP-1 was markedly enhanced, whereas TIMP-1 was decreased. 4. These results showed that I3C protected PCLS against alcoholic liver injury, which might be associated with the regulation of ethanol metabolic enzymes, attenuation of oxidative injury and acceleration of collagen degradation.

Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies.

Activation of hepatic stellate cells (HSCs) is the dominant event in liver fibrosis. The early events in the organization of HSC activation have been termed initiation. Initiation encompasses rapid changes in gene expression and phenotype that render the cells responsive to cytokines and other local stimuli. Cellular responses following initiation are termed perpetuation, which encompasses those cellular events that amplify the activated phenotype through enhanced growth factor expression and responsiveness. Multiple cells and cytokines play a part in the regulation of HSC activation. HSC activation consists of discrete phenotype responses, mainly proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis and retinoid loss. Currently, antifibrotic therapeutic strategies include inhibition of HSC proliferation or stimulation of HSC apoptosis, downregulation of collagen production or promotion of its degradation, administration of cytokines, and infusion of mesenchymal stem cells. In this review, we summarize the latest advances in our understanding of the mechanisms of HSC activation and possible antifibrotic therapeutic strategies.

Nicotine-induced prenatal overexposure to maternal glucocorticoid and intrauterine growth retardation in rat.

Overexposure to glucocorticoid during fetal development can result in intrauterine growth retardation (IUGR) as well as other diseases after birth. The purpose of this study is to investigate the possibility of glucocorticoid disturbance-mediated nicotine-induced IUGR after chronic prenatal exposure. Nicotine at 1.0mg/kg twice a day was administered subcutaneously to pregnant rats from gestational day (GD) 8 to GD 15 (mid-gestation) or GD 21 (late-gestation). Placental weights and fetal developmental parameters were recorded. Corticosterone levels were determined by radioimmunoassay. The mRNA expressions of adrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD-2) were determined using real-time quantitative RT-PCR. The results showed that prenatal chronic nicotine exposure causes IUGR in rats (P<0.01); in response to nicotine exposure, maternal serum corticosterone levels were elevated at mid- and late-gestations (P<0.05); mRNA expressions of StAR and P450scc increased in maternal adrenals (P<0.05 or 0.01) but decreased in fetal adrenals (P=0.16 or 0.11). Furthermore, the mRNA levels of placental 11 beta-HSD-2 were reduced at mid- and late-gestations (P<0.05). These results suggest that nicotine-induced IUGR is associated with the disturbances of glucocorticoid homeostasis in maternal and fetal rats. A possible underlying mechanism is that long term nicotine administration leads to fetal overexposure to maternal glucocorticoid by the combined effect of increased maternal glucocorticoid level and impaired placental barrier to it, all of which eventually leads to the fetal adrenocortical dysfunction and IUGR.

Study on the in vitro and in vivo activation of rat hepatic stellate cells by Raman spectroscopy.

The feasibility of a novel and efficient diagnostic method for liver fibrosis using Raman spectroscopy is studied. Confocal Raman spectroscopy (CRS) is utilized to monitor the molecular changes of hepatic stellate cells (HSCs) in vitro as well as in vivo activation. In vitro activation was induced by growth in uncoated plastic plates, while the in vivo activation is induced by a single intraperitoneal injection of carbon tetrachloride (CCl(4)). The biochemical changes of HSCs during activation such as the loss of retinoid, the increase of alpha-helical protein, and the increased production of extracellular matrix proteins are observed by CRS. A user-friendly autoclassifying system is also developed to classify Raman spectra of liver injury tissues with a 90% accuracy rate. Raman spectroscopy combined with a fiber optical probe could be potentially accomplished for in vivo detection, which can lead to a novel and efficient diagnosis for liver fibrosis.

Protective effect of verapamil on multiple hepatotoxic factors-induced liver fibrosis in rats.

The purpose of the present work was to investigate the effect of verapamil on liver fibrosis induced by multiple hepatotoxic factors in rats. Male Wistar rats were divided into a normal control group, a liver fibrosis model control group, and verapamil groups with different dosages. Multiple hepatotoxic factors including carbon tetrachloride (CCl(4)), ethanol and high cholesterol were used to make the animal model of liver fibrosis. The parameters of serum l-alanine aminotransferase (ALT), liver malondialdehyde and hydroxyproline contents were measured. Samples of the liver obtained by biopsy were subjected to histological and immunohistochemical studies for the expressions of alpha-smooth muscle actin (alpha-SMA) and transforming growth factor-beta(1) (TGF-beta(1)). Results showed that verapamil induced a dose-dependent decrease of serum ALT, liver malondialdehyde and hydroxyproline compared with liver fibrosis model control. Verapamil reduced hepatocyte degeneration and necrosis, and delayed the formation of liver fibrosis. The levels of expression of alpha-SMA and TGF-beta(1) in the hepatic tissue of three of the verapamil-treated groups were significantly less than those of the liver fibrosis model control group. The results showed that verapamil acts against the formation of liver fibrosis, the mechanism might be due to a protective effect for hepatocytes and through decreasing TGF-beta(1) to block the activation of hepatic stellate cells (HSCs) and collagen gene expression.

Effects of hepatic fibrosis on ofloxacin pharmacokinetics in rats.

The purpose of the present work was to study the pharmacokinetics of ofloxacin, a poorly metabolised drug, in experimental hepatic fibrosis. The possible roles of small intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP) in the bioavailability of ofloxacin were also evaluated. Rat hepatic fibrosis model was successfully induced using complex factors including carbon tetrachloride, ethanol and high fat. After rats received a single oral or intravenous dose of ofloxacin (40 mg kg(-1)), the plasma concentrations of ofloxacin were monitored at the scheduled time using spectrofluorimetric assay. Plasma concentration-time profiles were comodeled using compartmental method. Meanwhile, microsomal CYP isoenzymatic levels and P-gp expression in small intestines were compared between normal and hepatic fibrosis rats. When ofloxacin was administered intravenously, C(max) and the distribution half-life increased significantly in comparison with normal group, whereas the distribution rate constants, the apparent volume of distribution decreased. Oral ofloxacin bioavailability was significantly altered in hepatic fibrosis rats. AUC and C(max) were reduced, while the absorption half-life, peak time and elimination half-life significantly were prolonged, suggesting that both the extent and the rate of ofloxacin absorption were decreased. Furthermore, the increases in the levels of microsomal ethoxyresorufin O-deethylase and erythromycin N-demethylase were accompanied with up-regulation of mdr 1a mRNA in the small intestines of hepatic fibrosis rats when compared to those of the normal rats. The Results showed that pharmacokinetics of ofloxacin could be altered in hepatic fibrosis. Up-regulated P-gp expression and increased CYP isoenzymatic activities of small intestines in hepatic fibrosis rats may contribute to the decreased bioavailability and increased elimination of ofloxacin after oral administration.

[Antagonistic effect of sodium ferulate on glycerol-induced renal oxidative injury in mice].

AIM: To investigate the effect of sodium ferulate (SF) on glycerol-induced renal injury. METHODS: Glycerol solution 50% was injected intramuscularly to establish a model of acute tubular necrosis in mice. SF was administered intraperitoneally at the dose of 100-200 mg.kg-1 at the beginning of establishing the model and its effect was observed by monitoring renal function, antioxidative functions and renal pathologic histology. RESULTS: At 6 and 72 h after glycerol injection, SF treatment (100-200 mg.kg-1) showed significant and dose-dependent antagonistic actions on the increment of blood urea nitrogen (BUN), creatinine (Cr), and N-acetyl-beta-glucosaminidase (NAG) induced by glycerol. The increase of renal malondialdehyde (MDA) content and the decrease of glutathione content, glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), catalase (Cat) and superoxide dismutase (SOD) activities resulting from glycerol injection were remarkably inversed by SF at the dose of 200 mg.kg-1. Meanwhile, improvement of the renal histology was observed as well. CONCLUSION: SF showed beneficial effect on glycerol-induced acute tubular necrosis due to its antioxidative action.