Tatsuya Kanto

Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma

Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease‐stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved self‐digestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3‐I to LC3‐II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3‐II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP‐GFP‐LC3 fluorescence change in sorafenib‐treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3‐II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA‐mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.

Bak deficiency inhibits liver carcinogenesis: a causal link between apoptosis and carcinogenesis.

BACKGROUND & AIMS Hepatocyte apoptosis is a key feature of chronic liver disease including viral hepatitis and steatohepatitis. A previous study demonstrated that absence of the Bcl-2 family protein Mcl-1 led to increased hepatocyte apoptosis and development of liver tumors in mice. Since Mcl-1 not only inhibits the mitochondrial pathway of apoptosis but can also inhibit cell cycle progression and promote DNA repair, it remains to be proven whether the tumor suppressive effects of Mcl-1 are mediated by prevention of apoptosis. METHODS We examined liver tumor development, fibrogenesis, and oxidative stress in livers of hepatocyte-specific knockout (KO) of Mcl-1 or Bcl-xL, another key antagonist of apoptosis in hepatocytes. We also examined the impact of additional KO of Bak, a downstream molecule of Mcl-1 towards apoptosis but not the cell cycle or DNA damage pathway, on tumor development, hepatocyte apoptosis, and inflammation. RESULTS Bcl-xL KO led to a high incidence of liver tumors in 1.5-year-old mice, similar to Mcl-1 KO. Bcl-xL- or Mcl-1-deficient livers showed higher levels of TNF-α production and oxidative stress than wild-type livers at as early as 6 weeks of age and oxidative DNA damage at 1.5 years. Deletion of Bak significantly inhibited hepatocyte apoptosis in Mcl-1 KO mice and reduced the incidence of liver cancer, coinciding with reduction of TNF-α production, oxidative stress, and oxidative DNA damage in non-cancerous livers. CONCLUSIONS Our findings strongly suggest that chronically increased apoptosis in hepatocytes is carcinogenic and offer genetic evidence that inhibition of apoptosis may suppress liver carcinogenesis in chronic liver disease.

Absence of invariant natural killer T cells deteriorates liver inflammation and fibrosis in mice fed high-fat diet

BackgroundInvariant natural killer T (iNKT) cells have been suggested to play critical roles in a wide range of immune responses by acting in a proinflammatory or anti-inflammatory manner. Nonalcoholic steatohepatitis (NASH) is a chronic liver disease progressing to advanced cirrhosis and hepatocellular carcinoma. Despite the abundance of iNKT cells in the liver, their role in the pathogenesis of NASH remains obscure. Here, we investigated their role in the development of diet-induced steatosis/steatohepatitis.MethodsWe used BALB/c wild-type mice and Jα18-deficient (KO) mice lacking iNKT cells fed either a normal diet or a high-fat diet (HFD). The liver and blood were collected from these mice to examine liver inflammation, steatosis, and fibrosis at the indicated time points.ResultsKO mice fed the HFD, compared with control mice fed the HFD, exhibited a clearly higher serum alanine aminotransferase level and a greater number of hepatic inflammatory foci, although there was no significant difference in hepatic lipid retention between these groups of mice. The HFD enhanced hepatic messenger RNA expression of inflammatory cytokines and chemokines in KO but not in control mice. The HFD also increased the proportion of hepatic CD4 T cells and CD8 T cells that composed hepatic inflammatory foci in KO mice, but not in the controls. Prolonged feeding with the HFD augmented liver fibrosis in KO but not in control mice.ConclusionsThese findings indicate that iNKT cells play a protective role against liver inflammation progressing to fibrosis, but not against steatosis, enhanced by dietary excess fat, suggesting a key role of these cells in NASH pathogenesis.

Fibroblast growth factor‐2 enhances NK sensitivity of hepatocellular carcinoma cells

The roles of fibroblast growth factor‐2 (FGF‐2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF‐2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF‐2 in NK sensitivity of HCC cells. The FGF‐2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF‐2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF‐2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF‐2 was significantly lower than that with low levels of serum FGF‐2. Proinflammatory cytokines, such as IL‐1β and IL‐6, induced FGF‐2 expressions in HCC cells and normal hepatocytes. FGF‐2 stimulation resulted in increasing the expression of the membrane‐bound major histocompatibility complex class I‐related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti‐FGF receptor‐2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF‐2 stimulated HCC cells. FGF‐2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF‐2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.

Involvement of STAT3-regulated hepatic soluble factors in attenuation of stellate cell activity and liver fibrogenesis in mice.

Glycoprotein 130 (gp130)/signal transducer and activator of transcription 3 (STAT3) signaling in hepatocytes controls a variety of physiological and pathological processes including liver regeneration, apoptosis resistance and metabolism. Recent research has shed light on the importance of acute phase proteins (APPs) regulated by hepatic gp130/STAT3 in host defense through suppression of innate immune responses during systemic inflammation. To examine whether these STAT3-regulated soluble factors directly affect liver fibrogenic responses during liver injury, hepatocyte-specific STAT3 knockout (L-STAT3 KO) mice and control littermates were subjected to bile duct ligation (BDL) and examined 10 days later. In contrast to controls, L-STAT3 KO mice failed to produce APPs, such as serum amyloid A and haptoglobin, after BDL. Whereas L-STAT3 KO mice displayed similar levels of cholestasis, inflammatory cell infiltration and regeneration in the liver, they developed exacerbated liver injury and fibrosis with significant increases in expression of alpha-smooth muscle actin and type I collagen genes. In vitro experiments revealed that attenuated expression of APPs in primary hepatocytes isolated from L-STAT3 KO mice with IL-6 exposure, compared to wild-type hepatocytes. The cultured supernatant from IL-6-treated wild-type hepatocytes inhibited expression of alpha-smooth muscle actin and type I collagen genes in activated hepatic stellate cells (HSCs), whereas this did not occur with the supernatant from IL-6-treated knockout hepatocytes or with control medium. In conclusion, the absence of STAT3 in hepatocytes leads to exacerbation of liver fibrosis during cholestasis. Soluble factors released from hepatocytes, dependent on STAT3, collectively play a protective role in liver fibrogenesis through an inhibitory effect on activated HSCs.

Comparative analyses of regulatory T cell subsets in patients with hepatocellular carcinoma: A crucial role of CD25−FOXP3− T cells

Regulatory T cells (Tregs) play pivotal role in cancer‐induced immunoediting. Increment of CD25high+FOXP3+ natural Tregs has been reported in patients with hepatocellular carcinoma (HCC); however, the involvement of other type of Tregs remain elusive. We aimed to clarify whether FOXP3− Tregs are increased and functionally suppressive or not in patients with HCC. We enrolled 184 hepatitis C‐infected patients with chronic liver diseases or HCC, 57 healthy subjects and 27 HCC patients with other etiology. Distinct Treg subsets were phenotypically identified by the expression of CD4, CD25, CD127 and forkhead/winged helix transcription factor (FOXP3). Their gene profiles, frequency and suppressor functions against T cell proliferation were compared among the subjects. To examine the molecules involving in Treg differentiation, we cultured naive CD4+ T cells in the presence of HCC cells and dendritic cells. We determined two types of CD4+CD127− T cells with comparable regulatory ability; one is CD25high+ cells expressing FOXP3 (CD25high+FOXP3+ Tregs) and the other is CD25− cells without FOXP3− expression (CD25−FOXP3− cells). The peripheral or intrahepatic frequency of CD25−FOXP3− Tregs in HCC patients is higher than those in other groups, of which significance is more than CD25high+FOXP3+ cells. Of importance, CD25−FOXP3− Tregs, but not CD25high+FOXP3+ cells, dynamically change in patients accompanied by the ablation or the recurrence of HCC. CD25−FOXP3− T cells with CD127−IL‐10+ phenotype are inducible in vitro from naive CD4+ T cells, in which programmed cell death 1 ligand 1, immunoglobulin‐like transcript 4 and human leukocyte antigen G are involved.. In conclusion, CD25−FOXP3− Tregs with suppressive capacity are increased in patients with HCC, suggesting their distinct roles from CD25+FOXP3+ Tregs.

Interferon-α suppresses hepatitis B virus enhancer II activity via the protein kinase C pathway.

HBV has two enhancer (En) regions each of which promotes its own transcription. En II regulates production of pregenomic RNA, a key product of HBV replication, more strongly than En I. Although IFN-α has been found to suppress En I activity, its effect on En II activity has not been examined. Here we used luciferase assay to demonstrate that IFN-α suppresses En II activity. Analysis with several deletion/mutation constructs identified two major segments, nt 1703-1727 and nt 1746-1770, within the En II sequence as being responsible for the suppressive effects of IFN-α. Pre-treatment with protein kinase C (PKC) inhibitors blocked this effect regardless of the expression levels of phospho-STAT1 and Mx upon IFN-α stimulation. These results indicate that IFN-α suppresses En II activity via the PKC pathway, which may be an alternative suppressive pathway for HBV replication. (136 words).

The combination therapy of α-galactosylceramide and 5-fluorouracil showed antitumor effect synergistically against liver tumor in mice

α‐Galactosylceramide (α‐GalCer) has been reported to be therapeutic against metastatic liver tumors in mice. However, little is known regarding the efficacy of combined chemo‐immunotherapy using α‐GalCer and anticancer drugs. In this study, we evaluated the antitumor effect of the combination therapy of α‐GalCer and 5‐fluorouracil (5‐FU) against liver tumors of MC38 colon cancer cells. The liver weights of tumor‐bearing mice treated with the combination were significantly lower than those of nontreated mice and of mice treated with 5‐FU or α‐GalCer alone. No toxic effects on the liver and renal functions were observed in any of the treatment groups. α‐GalCer treatment induced significant activation of liver NK cells in vivo, but 5‐FU treatment did not. 5‐FU treatment resulted in a significant upregulation of NKG2D activating molecules (Rae‐1 and H60) and DNAM‐1 ligands (CD112 and CD155) on MC38 cells, but α‐GalCer did not. The cytolytic activity of α‐GalCer‐activated liver mononuclear cells against 5‐FU‐treated MC38 cells was significantly higher than that against nontreated cells. The increase of the cytolytic activity induced by 5‐FU partially depended on NKG2D‐Rae‐1 or H60 signals. Depletion of NK cells significantly inhibited the antitumor efficacy of 5‐FU against MC38 liver tumors, which suggested that the antitumor effect of 5‐FU partially depended on the cytolytic activity of NK cells. These results demonstrated that the combination therapy of α‐GalCer and 5‐FU produced synergistic antitumor effects against liver tumors by increasing the expression of NK activating molecules on cancer cells. This study suggests a promising new chemo‐immunotherapy against metastatic liver cancer.

Interleukin-1β enhances the production of soluble MICA in human hepatocellular carcinoma

The production of soluble major histocompatibility complex class I–related chain A (MICA) is thought to antagonize NKG2D-mediated immunosurveillance. Interleukin-1β (IL-1β) is elevated in patients with chronic hepatitis C (CH), and this might contribute to the escape of hepatocellular carcinoma (HCC) cells from innate immunity. In this study, we investigated the immunoregulatory role of IL-1β in the production of soluble MICA of HCC cells. First, we investigated the correlation between the serum IL-1β levels and soluble MICA in CH patients. Serum IL-1β levels were associated with soluble MICA levels in CH patients. The serum IL-1β levels of CH patients with the HCC occurrence were significantly higher than those of CH patients without HCC. We next examined the MICA production of IL-1β-treated HCC cells. Addition of IL-1β resulted in significant increase in the production of soluble MICA in HepG2 and PLC/PRF/5 cells, human HCC cells. But soluble MICA was not detected in both non-treated and IL-1β-treated normal hepatocytes. Addition of IL-1β did not increase the expressions of membrane-bound MICA on HCC cells. These were observed similarly in various cancer cells including a gastric cancer (MKN1), two colon cancers (HCT116 and HT29) and a cervical cancer (HeLa). Addition of IL-1β also increased the expression of a disintegrin and metalloproteinase (ADAM)9 in HCC cells, and the knockdown of ADAM9 in IL-1β-treated HCC cells resulted in the decrease in the production of soluble MICA of HCC cells. These findings indicate that IL-1β might enhance the production of soluble MICA by activating ADAM9 in human HCC.