An interim safety analysis of hepatocellular carcinoma patients administrating oral vitamin K with or without sorafenib.

BACKGROUNDS/AIMS: Vitamin K may plays a role in controlling hepatocellular carcinoma (HCC) cell growth. In this study, we intended to present 5-year experience of 72 patients receiving oral vitamin K with or without sorafenib. Its end-point was to evaluate the safety of combination therapy using sorafenib and vitamin K. METHODS: An interim analysis was performed as a single-arm cross-sectional study, including 72 HCC patients who underwent liver resection or transplantation and administered oral vitamin K2 alone (n=47) or with sorafenib (n=25). RESULTS: In all patients, administration of vitamin K2 analog 45 mg/day did not show any noticeable adverse side-effect during vitamin K therapy of 23.3±10.6 months, except for one patient who experienced skin rash at the third day of vitamin K therapy. In 25 patients receiving sorafenib and vitamin K for 6 months or longer, any noticeable adverse side-effect suspected of vitamin K origin was not identified yet. A small proportion of patients showed unexpectedly favorable anti-tumor effects after use of vitamin K with or without sorafenib. CONCLUSIONS: Because add-on of oral vitamin K did not increase the adverse side-effects of sorafenib, a combination therapy with these two agents appears to be worthy of further clinical trial with an expectation of synergistic therapeutic effects.

Sorafenib inhibits migration and invasion of hepatocellular carcinoma cells through suppression of matrix metalloproteinase expression.

Sorafenib increases survival of patients with advanced hepatocellular carcinoma (HCC) by inhibiting RAF kinase and receptor tyrosine kinase activity, but involvement of sorafenib in fibrosis and epithelial-mesenchymal transition (EMT) remains unclear. To elucidate effects of sorafenib on EMT progression and matrix metalloproteinase (MMP) activity, levels of E-cadherin, N-cadherin, and MMPs were evaluated in HepG2 human HCC cells induced by hepatocyte growth factor (HGF). Scratching cell migration assay, matrigel cell invasion assay, and immuno histochemistry were performed to examine effects of sorafenib on tumor metastasis and MMP expression. Sorafenib inhibited HGF-induced EMT and suppressed cell migration and invasion. Treatment with sorafenib significantly reduced HGF-enhanced expression of MMPs, suggesting that inhibition of MMP activity contributes to suppression of cellular motility and invasiveness of HepG2 cells. Neutralization of MMP activity by antibodies to MMP2/9, broad-spectrum MMP inhibitor or selective gelatinase inhibitor resulted in significant suppression of HGF-induced EMT and cell migration/invasion. Sorafenib treatment and MMP inactivation inhibited HGF-induced c-MET and MEK/ERK pathways. Sorafenib reduced MMP activity in this HGF-induced tumorigenic model of HCC. These findings provide in vitro evidence that sorafenib suppresses HGF-induced EMT and cell migration/invasion, as well as HGF-induced c-MET and MEK/ERK pathways.

Synergistic effect of sorafenib and vitamin K on suppression of hepatocellular carcinoma cell migration and metastasis.

Vitamin K plays a role in controlling cell growth. Anti-angiogenic effects of sorafenib lead to impairment of vitamin K uptake and induction of des-γ-carboxyprothrombin release by hepatocellular carcinoma (HCC) cells. We examined sorafenib and vitamin K individually and in combination regarding their ability to suppress migration and metastatic potential of HCC cells. HepG2 cells (HCC cell line) were treated with hepatocyte growth factor (HGF). E-Cadherin expression, phospho-MET (p-MET), and phospho-extracellular signal-regulated kinase (p-ERK) levels and cell migration were evaluated. HGF-stimulated HepG2 cells, which were treated with a combination of sorafenib and vitamin K, showed significantly increased expression of E-cadherin and impairment of migration ability compared to when treated with either agent alone. This combination therapy also induced marked inhibition of epithelial-mesenchymal transition phenotype; inhibition of HGF-stimulated cell proliferation, invasion and migration; and inhibition of HGF/c-MET signaling pathway. Levels of p-MET and p-ERK were also significantly reduced by this combination. Our experimental study demonstrated that sorafenib and vitamin K can function synergistically to inhibit the migration and proliferation of HCC cells. Combination therapy with sorafenib and vitamin K appears to be worthy of clinical trial with expectation of synergistic therapeutic effects.

Therapeutic potentials occurring during the early differentiation process of mesenchymal stem cells in a rats model with thioacetamide-induced liver fibrosis.

BACKGROUNDS/AIMS: Mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, The purpose of this study is to investigate the MSCs' differentiation process and therapeutic potentials by comparing isolated MSCs with HGF-treated MSCs in rat's model with thiacetamide (TAA)-induced cirrhosis. METHODS: Male Sprague-Dawley (SD) rats, weighing 100-150 g were used in this study. To induce liver fibrosis, recipient rats were taken with 0.04% thioacetamide (TAA) in the drinking water (400 mg TAA/L) for 8 weeks. The rats underlying liver cirrhosis were divided into 3 groups according to the transplanted materials, compared to normal saline as control (I) and isolated MSCs (II) HGF-treated MSCs. RESULTS: Severe hepatic fibrosis and hepatocyte destruction were detected in the control group. Less hepatic cirrhosis and collagen formation, more hepatocyte regeneration and glycogen storage were detected in isolated MSCs compared to HGF-treated MSCs group, Distribution of red autofluorescence is mainly localized near the sinusoids in isolated MSCs, scattered away the sinusoids in HGF-treated MSCs group. MSCs transdifferentiated into CK-19 postive Oval cells and then to albulmin-producing hepatocytes, HGF treated MSCs differentiated into hepatocyte without the intermediate oval cells phase. HGF treated MSCs became the CK18-positive, MSCs became CD 90-positive. CONCLUSIONS: Significant hepatocyte differentiation occurred in not HGF-treated MSCs but isolated MSCs group unexpectedly. These results suggest that the beneficial effect of MSCs on in rat's model with TAA-induced cirrhosis may occur during early differentiation course of MSCs. Mature hepatocyte itself has a little effect on the accelerated differentiation and functional capacity of hepatic lineage cell-line.

Preoperative peripheral blood human telomerase reverse transcriptase mRNA concentration is not a prognostic factor for resection of hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) often recurs after complete surgical resection. Detection of markers of residual circulating cancer cells may predict postoperative HCC recurrence. Human telomerase reverse transcriptase (hTERT) mRNA may be a candidate tumor marker. METHODOLOGY: We prospectively assessed the expression patterns and prognostic value of preoperative peripheral blood hTERT mRNA in patients with HCC undergoing hepatic resection (n=17) or liver transplantation (n=6). As controls, we assessed hTERT mRNA in patients with liver cirrhosis without HCC (n=6) and in living liver donors (n=4). Concentrations of hTERT mRNA were measured by real-time quantitative reverse transcription polymerase chain reaction (RTPCR). RESULTS: No significant difference was observed in the levels of hTERT mRNA between the HCC and control groups. Only alpha-fetoprotein ≥400ng/mL was associated with greater expression levels of hTERT mRNA. At a median follow-up of 30 months, HCC recurred in 10 of 17 resected patients, but in none of the 6 liver transplant recipients. hTERT mRNA concentration was not associated with HCC recurrence after either resection or liver transplantation. CONCLUSIONS: Peripheral blood hTERT mRNA concentration is not a likely marker for the diagnosis or prognosis of HCC, especially in patients undergoing resection. Owing to the small number of transplanted patients assessed, the clinical significance of hTERT mRNA concentration was not objectively verified, suggesting the need for a study in larger numbers of HCC patients undergoing liver transplantation.

Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis.

Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.

Sequential preoperative ipsilateral hepatic vein embolization after portal vein embolization to induce further liver regeneration in patients with hepatobiliary malignancy.

OBJECTIVE: To assess the effect of ipsilateral hepatic vein embolization (HVE) performed after portal vein embolization (PVE) on liver regeneration. SUMMARY BACKGROUND DATA: PVE induces shrinkage of the embolized lobe and compensatory enlargement of the non-embolized lobe, but it does not always induce sufficient liver regeneration. There was no effective method to accelerate liver regeneration in addition to PVE yet. METHODS: During a 1-year study period, preoperative HVE were performed on 12 patients who had shown limited liver regeneration after PVE awaiting right hepatectomy. The right hepatic vein was embolized with multiple coils after insertion of vena cava filters or vascular plugs. RESULTS: No HVE procedure-related complications occurred, but embolization of the wrong hepatic vein trunk occurred in 1 patient. The increase in blood liver enzymes after HVE was comparable with that after PVE alone. In 9 patients who underwent hepatectomy, the proportions of future liver remnant volume to total liver volume were 34.8% +/- 1.5% before PVE, 39.7% +/- 0.6% 1 to 2 weeks after PVE, 44.2% +/- 1.1% 2 weeks after HVE, and 64.5% +/- 6.2% 1 week after right hepatectomy. Cirrhotic livers showed lower regeneration rates following HVE after PVE and 1 patient underwent hepatectomy 17 months after HVE. Immunohistochemistry showed that apoptosis occurred more in the liver area affected by both PVE and HVE than in that affected by PVE alone. CONCLUSIONS: Preoperative sequential application of PVE and HVE seems to be safe and effective in facilitating contralateral liver regeneration by inducing more severe liver damage than PVE alone.

Inhibitory effects of gallic acid and quercetin on UDP-glucose dehydrogenase activity.

We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

Protective effects of ascorbic acid against lead-induced apoptotic neurodegeneration in the developing rat hippocampus in vivo.

Lead is a neurotoxin that affects the developing central nervous system and may potentially induce apoptotic cell death. We investigated the effect of ascorbic acid against lead-induced neurotoxicity in the developing rat hippocampus. Female Sprague-Dawley rats were divided into three groups: control group, lead-treated group and lead plus ascorbic acid-treated group. Lead (0.2% lead acetate) was administered to female rats during pregnancy and lactation, in their drinking water. During this period, rats in the lead plus ascorbic acid-treated group received 100 mg/kg/day ascorbic acid, orally. At the end of the treatment, neuronal damage, apoptosis and blood lead levels were determined and the levels of Bax and Bcl-2 were immunodetected in the hippocampus of 21-day-old male pups. Histopathological evaluation demonstrated that ascorbic acid significantly attenuates apoptosis in the developing hippocampus and also spares hippocampal CA1, CA3 and dentate gyrus (DG) neurons. Simultaneous administration of ascorbic acid and lead lowered the level of Bax protein and increased Bcl-2 in pup hippocampus and reduced lead level in blood of dams compared with lead-treated only. Based on these results, it seems that ascorbic acid may potentially be beneficial in treating lead-induced brain injury in the developing rat brain.

Alteration of the quaternary structure of human UDP-glucose dehydrogenase by a double mutation.

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.

Muscle regeneration by adipose tissue-derived adult stem cells attached to injectable PLGA spheres.

The [corrected] use of adult stem cells for cell-based tissue engineering and regeneration strategies represents a promising approach for skeletal muscle repair. We have evaluated the combination of adipose tissue-derived adult stem cells (ADSCs) obtained from autologous liposuction and injectable poly(lactic-co-glycolic acid) (PLGA) spheres for muscle regeneration. ADSCs attached to PLGA spheres and PLGA spheres alone were cultured in myogenic medium for 21 days and injected subcutaneously into the necks of nude mice. After 30 and 60 days, the mice were sacrificed, and newly formed tissues were analyzed by immunostaining, H and E staining, and RT-PCR. We found that ADSCs attached to PLGA spheres, but not PLGA spheres alone, were able to generate muscle tissue. These findings suggest that ADSCs and PLGA spheres are useful materials for muscle tissue engineering and that their combination can be used in clinical settings for muscle regeneration.

Characterization of nonpolio enteroviruses recovered from patients with aseptic meningitis in Korea.

OBJECTIVES: We attempted to characterize nonpolio enteroviruses recovered from Korean patients with aseptic meningitis. METHODS: We performed RT-PCR on the 5'-nontranslated region using clinical specimens. Infectious clinical isolates were amplified by infecting Vero cells with RT-PCR-positive clinical specimens. We then investigated the direct effect in primary neuronal cells or cardiomyocytes following virus infection. RESULTS: Total 12 clinical isolates were subtypically analyzed by both RT-PCR/sequencing comparison of the VP-1 region and neutralization assay. 43-2, 43-2S, 57 and 58 were found to be coxsackievirus B1 (CVB1), 312 to be CVB5, 14-2S and 327 to be echovirus 6, 165 to be echovirus 9, 337 to be echovirus 11, and 270 to be echovirus 30. All the clinical isolates tested showed profound cytotoxicity to various degrees in the primary neuronal cells within 24 h postinfection at 10 MOI. By contrast, a significant cytopathic effect was observed in the primary cardiomyocytes at 3-5 days postinfection at 50 MOI. CONCLUSIONS: The present study suggests that the clinical isolates recovered from Korean patients belonged to different CVB or echovirus serotypes and that these viruses showed diversities in their virulence in primary neuronal cells and cardiomyocytes.

Inhibitory effects of Cimicifuga heracleifolia extract on glutamate formation and glutamate dehydrogenase activity in cultured islets.

Hyperinsulinism-hyperammonemia syndrome is due either to hyperactivity of GDH or impaired inhibition of GDH by GTP. We have investigated the effect of Cimicifuga heracleifolia extract on the activities of glutamate dehydrogenase (GDH) in cultured rat islets. When the extract was present in the culture medium for 24 h prior to cell harvest, the Vmax of GDH was decreased by 45% with no significant change in Km. In addition, the concentration of alpha-ketoglutarate increased by approximately 39%, and glutamate decreased by 48%. Perfusion of islets with C. heracleifolia extract reduced insulin release by up to 47%. Although the relation between GDH activity and insulin release remains to be clarified, our results suggest that C. heracleifolia extract regulates insulin release by altering GDH activity in primary cultured islets and that this natural compound may be used to modulate GDH activity in patients with hyperinsulinism-hyperammonemia syndrome.

Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes.

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.

Differential activation of caspase-3 at two maturational stages during okadaic acid-induced rat neuronal death.

Okadaic acid (OA), a protein phosphatase inhibitor, is used as a research model of Alzheimer's disease to induce tau phosphorylation and neuronal death. We reported previously that OA induces neuronal apoptosis of immature neurons (in vitro days (IVD) 3-5), which is inhibited by cycloheximide (CHX). In this study, we demonstrate that CHX fails to prevent OA-induced neuronal death in mature neurons (IVD 14-15). Upon comparison of both types of dying cells, the immature neurons displayed characteristic features of apoptosis, such as nuclear fragmentation, phosphatidylserine externalization and prominent caspase-3 activation, while the mature neurons showed few characteristic features of apoptosis. Lack of the beneficial effects of CHX and the lesser activation of caspase-3 in the mature neurons argue against typical apoptotic neuronal death in the OA-induced neurodegeneration model.

Coxsackievirus B4-induced neuronal apoptosis in rat cortical cultures.

Enterovirus infections of the central nervous system (CNS) are common and important causes of morbidity in immunocompromised children and adults. In this study we identify and characterize coxsackievirus B4-induced neuronal death. To investigate the CNS pathophysiology resulting from this viral infection, cultured rat neurons were infected with coxsackievirus B4 (CVB4) and nuclear morphology, phosphatidylserine (PS) externalization, and the effects of Actinomycin D or cycloheximide (CHX) were examined. CVB4 induced neuronal cell death within 24 h while PS externalization was apparent in cell bodies 16 h after CVB4 infection. Actinomycin D or CHX significantly reduced CVB4 induced-neuronal cell death in a dose-dependent manner. Pretreatment with CHX or actinomycin D also inhibited nuclear condensation, which occurred after CVB4 infection. However, the changes were relatively unresponsive to zVAD-fmk. These results suggest that CVB4 induces CHX- and actinomycin D-sensitive, but zVAD-fmk-insensitive neuronal apoptosis.