Varying impact of lenvatinib or sorafenib therapy on skeletal muscle loss in patients with hepatocellular carcinoma.

BACKGROUND: Lenvatinib and sorafenib are key therapeutic agents for hepatocellular carcinoma. However, there are no useful biomarkers for selecting molecular-targeted agents (MTAs). Skeletal muscle volume is associated with the clinical outcomes in these patients. We investigated the effects of lenvatinib and sorafenib on the skeletal muscles of patients with HCC. METHODS: We evaluated the impact of skeletal muscle changes over a 3-month period for each MTA (n = 117; lenvatinib/sorafenib, 45/72). The skeletal muscle mass index (SMI) was measured at the third lumbar vertebra. Furthermore, we evaluated the direct effect of each MTA on primary human skeletal muscle cells by estimating muscle protein synthesis using western blot analysis. RESULTS: The median change in SMI was -0.7% (p = 0.959) and -5.9% (p <0.001) for the lenvatinib and sorafenib groups, respectively. Sorafenib had a greater effect on skeletal muscle loss than lenvatinib (p < 0.001). Additionally, SMI significantly decreased in the sorafenib group regardless of initial skeletal muscle volume (p < 0.001), whereas no significant differences were observed in the lenvatinib group. Sorafenib therapy (odds ratio [OR], 2.98; p = 0.023) and non-muscle depletion (OR, 3.31; p = 0.009) were associated with a decreased SMI. In vitro analysis showed that sorafenib negatively affected muscle synthesis compared to lenvatinib. CONCLUSIONS: Sorafenib may have a more negative effect on skeletal muscle than lenvatinib.

Regulation of Adenine Nucleotide Metabolism by Adenylate Kinase Isozymes: Physiological Roles and Diseases.

Adenylate kinase (AK) regulates adenine nucleotide metabolism and catalyzes the ATP + AMP ⇌ 2ADP reaction in a wide range of organisms and bacteria. AKs regulate adenine nucleotide ratios in different intracellular compartments and maintain the homeostasis of the intracellular nucleotide metabolism necessary for growth, differentiation, and motility. To date, nine isozymes have been identified and their functions have been analyzed. Moreover, the dynamics of the intracellular energy metabolism, diseases caused by AK mutations, the relationship with carcinogenesis, and circadian rhythms have recently been reported. This article summarizes the current knowledge regarding the physiological roles of AK isozymes in different diseases. In particular, this review focused on the symptoms caused by mutated AK isozymes in humans and phenotypic changes arising from altered gene expression in animal models. The future analysis of intracellular, extracellular, and intercellular energy metabolism with a focus on AK will aid in a wide range of new therapeutic approaches for various diseases, including cancer, lifestyle-related diseases, and aging.

Assessment of exposure and DNA damage from second-hand smoke using potential biomarker in urine: cigarettes and heated tobacco products.

Second-hand smoke exposure is an established cause of several adverse health effects. Tobacco smoke exposure in the environment has been improved by the WHO Framework Convention on Tobacco Control. However, concerns have been raised regarding the health effects of heated tobacco products. Analysis of tobacco smoke biomarkers is critical for assessing the health effects of second-hand tobacco smoke exposure. In this study, nicotine metabolites (nicotine, cotinine, trans-3'-hydroxycotinine) and carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were analysed in the urine of non-smokers with or without passive exposure to cigarettes and heated tobacco products. In addition, 7-methylguanine and 8-hydroxy-2'-deoxyguanosine were simultaneously measured as DNA damage markers. The results revealed higher levels of urinary nicotine metabolites and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in participants exposed to second-hand tobacco smoke (both cigarettes and heated tobacco products) at home. In addition, the urinary levels of 7-methylguanine and 8-hydroxy-2'-deoxyguanosine tended to be higher in the second-hand tobacco smoke-exposed group. The urinary levels of nicotine metabolites and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were high in workplaces with no protection against passive smoking. These biomarkers will be useful for evaluating passive exposure to tobacco products.

Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism.

The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.

Evaluation of the Effects of Microgravity on Activated Primary Human Hepatic Stellate Cells.

In recent years, research has been conducted to develop new medical treatments by simulating environments existing in space, such as zero-gravity. In this study, we evaluated the cell proliferation and gene expression of activated primary human hepatic stellate cells (HHSteCs) under simulated microgravity (SMG). Under SMG, cell proliferation was slower than in 1 G, and the evaluation of gene expression changes on day 1 of SMG by serial analysis of gene expression revealed the presence of Sirtuin, EIF2 signaling, hippo signaling, and epithelial adherence junction signaling. Moreover, reactive oxygen species were upregulated under SMG, and when N-acetyl-cystein was added, no difference in proliferation between SMG and 1 G was observed, suggesting that the oxidative stress generated by mitochondrial dysfunction caused a decrease in proliferation. Upstream regulators such as smad3, NFkB, and FN were activated, and cell-permeable inhibitors such as Ly294002 and U0126 were inhibited. Immunohistochemistry performed to evaluate cytoskeletal changes showed that more ß-actin was localized in the cortical layer under SMG.

MC180295 Inhibited Epstein-Barr Virus-Associated Gastric Carcinoma Cell Growth by Suppressing DNA Repair and the Cell Cycle.

DNA methylation of both viral and host DNA is one of the major mechanisms involved in the development of Epstein-Barr virus-associated gastric carcinoma (EBVaGC); thus, epigenetic treatment using demethylating agents would seem to be promising. We have verified the effect of MC180295, which was discovered by screening for demethylating agents. MC180295 inhibited cell growth of the EBVaGC cell lines YCCEL1 and SNU719 in a dose-dependent manner. In a cell cycle analysis, growth arrest and apoptosis were observed in both YCCEL1 and SNU719 cells treated with MC180295. MKN28 cells infected with EBV were sensitive to MC180295 and showed more significant inhibition of cell growth compared to controls without EBV infection. Serial analysis of gene expression analysis showed the expression of genes belonging to the role of BRCA1 in DNA damage response and cell cycle control chromosomal replication to be significantly reduced after MC180295 treatment. We confirmed with quantitative PCR that the expression levels of BRCA2, FANCM, RAD51, TOP2A, and CDC45 were significantly decreased by MC180295. LMP1 and BZLF1 are EBV genes with expression that is epigenetically regulated, and MC180295 could up-regulate their expression. In conclusion, MC180295 inhibited the growth of EBVaGC cells by suppressing DNA repair and the cell cycle.

Establishment of an Adult Medaka Fatty Liver Model by Administration of a Gubra-Amylin-Nonalcoholic Steatohepatitis Diet Containing High Levels of Palmitic Acid and Fructose.

Among lifestyle-related diseases, fatty liver is the most common liver disease. To date, mammalian models have been used to develop methods for inhibiting fatty liver progression; however, new, more efficient models are expected. This study investigated the creation of a new model to produce fatty liver more efficiently than the high-fat diet medaka model that has been used to date. We compared the GAN (Gubra-Amylin nonalcoholic steatohepatitis) diet, which has been used in recent years to induce fatty liver in mice, and the high-fat diet (HFD). Following administration of the diets for three months, enlarged livers and pronounced fat accumulation was noted. The GAN group had large fat vacuoles and lesions, including ballooning, compared to the HFD group. The GAN group had a higher incidence of lesions. When fenofibrate was administered to the fatty liver model created via GAN administration and liver steatosis was assessed, a reduction in liver fat deposition was observed, and this model was shown to be useful in drug evaluations involving fatty liver. The medaka fatty liver model administered with GAN will be useful in future fatty liver research.

Invasion inhibition in pancreatic cancer using the oral iron chelating agent deferasirox.

BACKGROUND: Iron is required for cellular metabolism, and rapidly proliferating cancer cells require more of this essential nutrient. Therefore, iron regulation may well represent a new avenue for cancer therapy. We have reported, through in vitro and in vivo research involving pancreatic cancer cell lines, that the internal-use, next-generation iron chelator deferasirox (DFX) exhibits concentration-dependent tumour-suppressive effects, among other effects. After performing a microarray analysis on the tumour grafts used in that research, we found that DFX may be able to suppress the cellular movement pathways of pancreatic cancer cells. In this study, we conducted in vitro analyses to evaluate the effects of DFX on the invasive and migratory abilities of pancreatic cancer cells. METHODS: We used pancreatic cancer cell lines (BxPC-3, Panc-1, and HPAF II) to examine the efficacy of DFX in preventing invasion in vitro, evaluated using scratch assays and Boyden chamber assays. In an effort to understand the mechanism of action whereby DFX suppresses tumour invasion and migration, we performed G-LISA to examine the activation of Cdc42 and Rac1 which are known for their involvement in cellular movement pathways. RESULTS: In our scratch assays, we observed that DFX-treated cells had significantly reduced invasive ability compared with that of control cells. Similarly, in our Boyden chamber assays, we observed that DFX-treated cells had significantly reduced migratory ability. After analysis of the Rho family of proteins, we observed a significant reduction in the activation of Cdc42 and Rac1 in DFX-treated cells. CONCLUSIONS: DFX can suppress the motility of cancer cells by reducing Cdc42 and Rac1 activation. Pancreatic cancers often have metastatic lesions, which means that use of DFX will suppress not only tumour proliferation but also tumour invasion, and we expect that this will lead to improved prognoses.

Metabolic Alterations in Spheroid-Cultured Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) play a vital role in liver fibrosis, and a greater understanding of their regulation is required. Recent studies have focused on relationships between extracellular matrix (ECM) stiffness and gene expression or cellular metabolism, but none have provided a detailed metabolic analysis of HSC changes in spheroid cultures. Accordingly, in the present study, we created an HSC spheroid culture and analyzed changes in gene expression and metabolism. Expression of α-smooth muscle actin (α-SMA) decreased in the spheroids, suppressing proliferation. Gene expression analysis revealed the cell cycle, sirtuin signaling, mitochondrial dysfunction, and the Hippo pathway to be canonical pathways, believed to result from decreased proliferative ability or mitochondrial suppression. In the Hippo pathway, nuclear translocation of the yes-associated protein (YAP) was decreased in the spheroid, which was associated with the stiffness of the ECM. Metabolome analysis showed glucose metabolism changes in the spheroid, including glutathione pathway upregulation and increased lipid synthesis. Addition of the glycolytic product phosphoenolpyruvate (PEP) led to increased spheroid size, with increased expression of proteins such as α-SMA and S6 ribosomal protein (RPS6) phosphorylation, which was attributed to decreased suppression of translation. The results of our study contribute to the understanding of metabolic changes in HSCs and the progression of hepatic fibrosis.

Bone marrow-derived humoral factors suppress oxidative phosphorylation, upregulate TSG-6, and improve therapeutic effects on liver injury of mesenchymal stem cells.

Mesenchymal stem cells, which have the potential to be used in regenerative medicine, require improvements in quality for patient use. To maintain stemness of cultured bone marrow-derived mesenchymal stem cells, we focused on the bone marrow microenvironment, generated a conditioned medium of whole bone marrow cells (BMC-CM), and assessed its effects on bone marrow-derived mesenchymal stem cells. BMC-CM suppressed morphological deterioration and proliferative decline in cultured bone marrow-derived mesenchymal stem cells, suppressed mitochondrial oxidative phosphorylation activity, a stemness indicator, and upregulated suppressors of oxidative phosphorylation such as hypoxia-inducible factor-1 alpha, Sirtuin 3, 4, and 5. Furthermore, BMC-CM upregulated TNF-stimulated gene 6 and ameliorated the therapeutic effects of cells on liver injury in carbon tetrachloride-administered rats. Since the elimination of 20-220-nm particles attenuated the effects of BMC-CM, we further analyzed exosomal microRNAs produced by whole bone marrow cells. Among the 49 microRNAs observed to be upregulated during the preparation of BMC-CM, several were identified that were associated with suppression of oxidative phosphorylation, upregulation of TNF-stimulated gene 6, and the pathogenesis of liver diseases. Thus, bone marrow-derived humoral factors including exosomal microRNAs may help to improve the therapeutic quality of bone marrow-derived mesenchymal stem cells for liver regenerative therapy.

Trans-portal hepatic infusion of cultured bone marrow-derived mesenchymal stem cells in a steatohepatitis murine model.

The incidence of nonalcoholic steatohepatitis-related liver cirrhosis is increasing. We used a steatohepatitis murine model fed a choline-deficient, l-amino acid-defined (CDAA) diet with a single injection of carbon tetrachloride (CCl4) to evaluate the efficacy of trans-portal hepatic infusion of bone marrow-derived mesenchymal stem cells (BMSCs) for liver fibrosis, liver steatosis, and oxidative stress. Mice were fed a CDAA diet and injected with a single intraperitoneal dose of CCl4 (0.5 ml/kg) after 4 weeks of CDAA diet. After 12 weeks of CDAA diet, 1 × 106 luciferase-positive syngeneic BMSCs (Luc-BMSCs) were infused into the animal spleen. An in vivo imaging system was used to confirm Luc-BMSC accumulation in the liver via the portal vein, and at 4 weeks after infusion, we compared liver fibrosis, liver steatosis, and oxidative stress. After the BMSC-infusion, serum albumin and serum total bilirubin were significantly improved. Liver fibrosis assessed by Sirius red staining, α-smooth muscle actin protein, and collagen 1A1 mRNA expression was significantly suppressed. Furthermore, liver steatosis area was significantly lower, the 8-hydroxy-2'-deoxyguanosine-positive cells were significantly fewer, and superoxide dismutase 2 protein expression of the liver was significantly increased. In conclusion, our data confirmed the efficacy of trans-portal hepatic infusion of BMSCs in a steatohepatitis murine model.

Analysis of Metabolomic Changes in Mesenchymal Stem Cells on Treatment with Desferrioxamine as a Hypoxia Mimetic Compared with Hypoxic Conditions.

Mesenchymal stem cells (MSCs) are commonly used in regenerative medicine, but their therapeutic effects vary depending on the culture environment. Hypoxic culturing can be used to maintain stem cells in an undifferentiated state, but is expensive and difficult to perform. The aim of this study was to determine the effectiveness of desferrioxamine (DFO), a hypoxia-mimetic reagent, as an alternative to hypoxic culturing by analyzing metabolic changes in MSCs under hypoxic conditions compared with changes induced by DFO. Low concentrations of DFO reduced mitochondrial activity and apoptosis. Therefore, low concentrations of DFO may be useful for MSC preconditioning. Metabolome analysis showed that both hypoxic treatment and DFO administration exhibited similar metabolite patterns except purine, pyrimidine, and tricarboxylic acid cycle (TCA) cycle related metabolites. Therefore, the use of DFO at low concentrations is a potential substitute for hypoxic culturing. These findings may form the foundation for the development of future regenerative therapies using MSCs. Stem Cells 2018;36:1226-1236.

NUPR1 acts as a pro-survival factor in human bone marrow-derived mesenchymal stem cells and is induced by the hypoxia mimetic reagent deferoxamine.

Differences in the culturing conditions of mesenchymal stem cells used in regenerative medicine may affect their differentiation ability, genome instability, and therapeutic effects. In particular, bone marrow-derived mesenchymal stem cells cultured under hypoxia are known to proliferate while maintaining an undifferentiated state and the use of deferoxamine, a hypoxia mimetic reagent, has proven to be a suitable strategy to maintain the cells under hypoxic metabolic state. Here, the deferoxamine effects were investigated in mesenchymal stem cells to gain insights into the mechanisms regulating stem cell survival. A 12-h deferoxamine treatment reduced proliferation, oxygen consumption, mitochondrial activity, and ATP production. Microarray analysis revealed that deferoxamine enhanced the transcription of genes involved in glycolysis and the HIF1α pathway. Among the earliest changes, transcriptional variations were observed in HIF1α, NUPR1, and EGLN, in line with previous reports showing that short deferoxamine treatments induce substantial changes in mesenchymal stem cells glycolysis pathway. NUPR1, which is induced by stress and involved in autophagy-mediated survival, was upregulated by deferoxamine in a concentration-dependent manner. Consistently, NUPR1 knockdown was found to reduce cell proliferation and increase the proapoptotic effect of staurosporine, suggesting that deferoxamine-induced NUPR1 promotes mesenchymal stem cell survival and cytoprotective autophagy. Our findings may substantially contribute to improve the effectiveness of mesenchymal stem cell-based regenerative medicine.

Mitochondrial Activity and Unfolded Protein Response are Required for Neutrophil Differentiation.

BACKGROUND/AIMS: Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in hematopoietic differentiation. However, the mechanistic linkage between ER stress/UPR and hematopoietic differentiation remains unclear. METHODS: We used bipotent HL-60 cells as an in vitro hematopoietic differentiation system to investigate the role of ER stress and UPR activity in neutrophil and macrophage differentiation. RESULTS: The in vitro differentiation analysis revealed that ER stress decreased during both neutrophil and macrophage differentiations, and the activities of PERK and ATF6 were decreased and that of IRE1α was increased during neutrophil differentiation in a stage-specific manner. By contrast, the activities of ATF6 and ATF4 decreased during macrophage differentiation. When the cells were treated with oligomycin, the expression of CD11b, a myelocytic differentiation marker, and morphological differentiation were suppressed, and XBP-1 activation was inhibited during neutrophil differentiation, whereas CD11b expression was maintained, and morphological differentiation was not obviously affected during macrophage differentiation. CONCLUSION: In this study, we demonstrated that neutrophil differentiation is regulated by ER stress/UPR that is supported by mitochondrial ATP supply, in which IRE1α-XBP1 activation is essential. Our findings provide the evidence that mitochondrial energy metabolism may play a critical role in neutrophil differentiation.

Effect of histidine on sorafenib-induced vascular damage: Analysis using novel medaka fish model.

BACKGROUND: Sorafenib (SFN) is an anti-angiogenic chemotherapeutic that prolongs survival of patients with hepatocellular carcinoma (HCC); its side effects, including vascular damages such as hand-foot syndrome (HFS), are a major cause of therapy discontinuation. We previously reported that maintenance of peripheral blood flow by intake of dried bonito broth (DBB) significantly prevented HFS and prolonged the administration period. The amino acids contained in DBB probably contribute to its effects, but the mechanism has not been clarified. We hypothesized that histidine, the largest component among the amino acids contained in DBB, has effects on SFN-induced vascular damage, and evaluated this possibility using a novel medaka fish model. METHODS: The fli::GFP transgenic medaka fish model has a fluorescently visible systemic vasculature. We fed the fish with SFN with and without histidine to compare blood flow and vascular structure among the differently fed models. The vascular cross-sectional area of each fish was measured to determine vascular diameter changes. RESULTS: Our results demonstrated that SFN-fed medaka developed a narrower vascular diameter. In addition, this narrowing was counteracted by addition of histidine to the medaka diet. We observed no positive effect of histidine on regeneration of cut vessels or on cell growth of endothelial cells and HCC cell lines. CONCLUSION: We proved the efficacy of the medaka model to assess vascular changes after administration of specific chemicals. And our results suggest that SFN causes vascular damage by narrowing peripheral vessel diameter, and that histidine effectively counteracts these changes to maintain blood flow.

Evaluating effects of L-carnitine on human bone-marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells showing potential for use in regenerative medicine. Culture techniques that are more stable and methods for the more efficient production of MSCs with therapeutic efficacy are needed. We evaluate the effects of growing bone marrow (Bm)-derived MSCs in the presence of L-carnitine, which is believed to promote lipid metabolism and to suppress apoptosis. The presence of L-carnitine decreased the degree of drug-induced apoptosis and suppressed adipogenic differentiation. Metabolomic analysis by means of the exhaustive investigation of metabolic products showed that, in addition to increased ß-oxidation and the expression of all carnitine derivatives other than deoxycarnitine (an intermediate in carnitine synthesis), polysaturated and polyunsaturated acids were down-regulated. An integrated analysis incorporating both serial analysis of gene expression and metabolomics revealed increases in cell survival, suggesting the utility of carnitine. The addition of carnitine elevated the oxygen consumption rate by BmMSCs that had been cultured for only a few generations and those that had become senescent following repeated replication indicating that mitochondrial activation occurred. Our exhaustive analysis of the effects of various carnitine metabolites thus suggests that the addition of L-carnitine to BmMSCs during expansion enables efficient cell production.

Effectiveness of tolvaptan monotherapy and low-dose furosemide/tolvaptan combination therapy for hepatoprotection and diuresis in a rat cirrhotic model.

Spironolactone and furosemide, which are used to treat ascites associated with decompensated cirrhosis, are ineffective in treating refractory ascites. Hence, combination therapy with tolvaptan, a vasopressin V2 receptor antagonist, has been approved in Japan. Tolvaptan monotherapy and combination therapy with furosemide inhibit fibrosis in cardiac remodeling; hence, we examined these therapies in a rat cirrhotic model, including their usefulness in inhibiting hepatic fibrosis. In the present study, we used a model of hepatic fibrosis induced by a choline-deficient l-amino-acid-defined diet + diethylnitrosamine. Rats were divided into a low-dose furosemide group (15 mg/kg/day), a high-dose furosemide group (100 mg/kg/day), a tolvaptan monotherapy group (10 mg/kg/day), a low-dose furosemide/tolvaptan combination therapy group, and a control group which received neither furosemide nor tolvaptan; we then assessed diuretic effects and hepatic fibrosis. The tolvaptan monotherapy group and the furosemide/tolvaptan combination therapy group demonstrated significantly higher urine volume than the control group and the low-dose furosemide group. In addition, tolvaptan monotherapy and low-dose furosemide/tolvaptan combination therapy were found to inhibit hepatic fibrosis and yield a hepatoprotective effect by an antioxidative mechanism. The results of the present study suggest that tolvaptan monotherapy and low-dose furosemide/tolvaptan combination therapy are highly effective for hepatoprotection and diuresis.