Masataka Okuno

Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma

Background. In patients with hepatocellular carcinoma (hepatoma), the rate of recurrent and second primary hepatomas is high despite surgical resection and percutaneous ethanol-injection therapy. We developed an acyclic retinoid, polyprenoic acid, that inhibits hepatocarcinogenesis in the laboratory and induces differentiation and apoptosis in cell lines derived from human hepatoma. In a randomized, controlled study, we tested whether the compound reduced the incidence of recurrent and second primary hepatomas after curative treatment. Methods. We prospectively studied 89 patients who were free of disease after surgical resection of a primary hepatoma or the percutaneous injection of ethanol. We randomly assigned the patients to receive either polyprenoic acid (600 mg daily) or placebo for 12 months. We studied the remnant liver by ultrasonography every three months after randomization. The primary end point of the study was the appearance of a histologically confirmed recurrent or new hepatoma. Results. Treatment with polyprenoic acid significantly reduced the incidence of recurrent or new hepatomas. After a median follow-up of 38 months, 12 patients in the polyprenoic acid group (27 percent) had recurrent or new hepatomas as compared with 22 patients in the placebo group (49 percent, P = 0.04). The most striking difference was in the groups that had second primary hepatomas-7 in the group receiving polyprenoic acid as compared with 20 in the placebo group (P=0.04 by the log-rank test). Cox proportional-hazards analysis demonstrated that as an independent factor, polyprenoic acid reduced the occurrence of second primary hepatomas (adjusted relative risk, 0.31 ; 95 percent confidence interval, 0.12 to 0.78). Conclusions. Oral polyprenoic acid prevents second primary hepatomas after surgical resection of the original tumor or the percutaneous injection of ethanol.

Synergistic effects of RXRα and PPARγ ligands to inhibit growth in human colon cancer cells—phosphorylated RXRα is a critical target for colon cancer management

Background and aims: The activation of the peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) that forms heterodimers with retinoid X receptors (RXRs) elicits an antineoplastic effect on colorectal cancer. It was previously reported that the accumulation of the non-functional phosphorylated form of RXR&agr; (p-RXR&agr;) interfered with its signalling and promoted the growth of hepatoma cells. In this study the effects of p-RXR&agr; on the ability of RXR&agr; and PPAR&ggr; ligands to inhibit growth in colon cancer cells was examined. Methods: The effects of the combination of the PPAR&ggr; ligand ciglitazone and the RXR&agr; lignad 9-cis-retinoic acid (RA) on inhibition of cell growth in Caco2 human colon cancer cells which express high levels of p-RXR&agr; protein were examined Results: The RXR&agr; protein was phospholylated and also accumulated in human colon cancer tissue samples as well as human colon cancer cell lines. When the phosphorylation of RXR&agr; was inhibited by the MEK inhibitor PD98059 or by transfection with a point-mutated RXR&agr;, which mimicked the unphosphorylated form, the combination of 9-cisRA and ciglitazone synergistically inhibited the cell growth and induced apoptosis. The combined treatment with these agents also caused a decrease in the expression levels of both cyclo-oxygenase-2 (COX-2) and c-Jun proteins and mRNAs. Reporter assays indicated that this combination induced the transcriptional activity of the peroxisome proliferator-responsive element promoter and also inhibited that of the AP-1 promoter. Conclusion: A malfunction of RXR&agr; due to phosphorylation is associated with colorectal cancer. Therefore, the inhibition of phosphorylation of RXR&agr; and the activation of the RXR–PPAR&ggr; heterodimer by their respective ligands may be useful in the chemoprevention and/or treatment of colorectal cancer.

Role of Transglutaminase 2 in Liver Injury via Cross-linking and Silencing of Transcription Factor Sp1

BACKGROUND & AIMS Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. METHODS Human hepatic cells or primary hepatocytes from rats or TG2+/+ and TG2-/- mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. RESULTS Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met. These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2+/+ mice, TG2-/- mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2+/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas-induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. CONCLUSIONS TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.

Prevention of Second Primary Tumors by an Acyclic Retinoid in Patients with Hepatocellular Carcinoma

Oral administration with acyclic retinoid, a synthetic vitamin A analog, for a limited period of 12 months (48 weeks) prevented the development of second primary hepatocellular carcinoma (HCC) and also improved the survival of patients who underwent curative treatments of the initial tumor. Following that randomized controlled study reported in 1996 and 1999, we have continued to follow up the patients by medical imaging and blood chemical analyses, and found that the preventive effect of acyclic retinoid lasted up to 199 weeks after randomization (or 151 weeks after completion of retinoid administration). The retinoid’s effect was not mediated by reduction in hepatic necro-inflammation since no significant decrease in serum aminotransferase activity was seen in the retinoid group. Such observation seems quite distinct from the cancer-preventive mechanism of interferon, a potent immunopreventive agent for HCC. We have also shown here the reduction by the retinoid in serum levels of lectin-reactive α-fetoprotein (AFP-L3) and protein induced by vitamin K absence or antagonist-II (PIVKA-II), both of which indicate the presence of latent HCC cells. These results suggest that acyclic retinoid may delete such malignant clones before they expand to clinically detectable tumors and thereby inhibited second primary HCC. Once such latent clones are eradicated, it may well take at least several years for the next cancer clone to arise clinically. This may possibly explain a reason for the long-term effect of the retinoid even after the limited period of administration.

9,13‐di‐cis‐Retinoic acid induces the production of tPA and activation of latent TGF‐β via RARα in a human liver stellate cell line, LI90

We studied the mechanism by which 9,13‐di‐cis‐retinoic acid (9,13dcRA), a novel and endogenous stereoisomer of all‐trans‐RA, induces TGF‐β formation in a human liver stellate cell line, LI90. 9,13dcRA induced the expression of RARα and RARβ, enhanced the production of tissue‐type plasminogen activator (tPA), thereby, surface plasmin levels, and induced the activation of latent TGF‐β. Similar effects were obtained with RARα‐selective retinoid, but not with RARβ‐ or RARγ‐selective retinoid, and the induction was inhibited by RARα‐selective antagonist. These results suggest that 9,13dcRA up‐regulates tPA expression, resulting in the formation of TGF‐β by LI90 cells, at least in part, via induction and activation of RARα.

Acyclic retinoid induces partial differentiation, down-regulates telomerase reverse transcriptase mRNA expression and telomerase activity, and induces apoptosis in human hepatoma-derived cell lines.

BACKGROUND/AIMS Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level. METHODS Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining. RESULTS Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis. CONCLUSIONS AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

Synergistic growth inhibition by acyclic retinoid and vitamin K2 in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K2 (VK2) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9‐cis retinoic acid (9cRA) plus VK2 in the HuH7 human HCC cell line. We found that the combination of 1.0 µM ACR or 1.0 µM 9cRA plus 10 µM VK2 synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK2 also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK2 alone inhibited phosphorylation of the retinoid X receptor (RXR)α protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal‐regulated kinase phosphorylation. Moreover, the inhibition of RXRα phosphorylation by VK2 was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK2 markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK2 cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRα and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC. (Cancer Sci 2007; 98: 431–437)

Chemoprevention of hepatocellular carcinoma: Concept, progress and perspectives

Abstract Hepatocellular carcinoma (HCC) often develops in patients with chronic liver diseases associated with hepatitis B (HBV) and hepatitis C (HCV) virus infections with high incidences. Particularly, post‐therapeutic recurrence encountered after the curative treatment of the preceding HCC may limit the prognosis. Thus, prevention of HCC is of great significance. In the present review, immunopreventions with α‐interferon and glycyrrhizin, as well as chemoprevention with acyclic retinoid, are discussed. α‐Interferon prevents the development of HCC not only in patients with a long‐term elimination of HCV (sustained virological responders), but in ones with normalized serum aminotransferases (sustained biochemical responders). Glycyrrhizin also suppresses serum aminotransferases and thereby prevents the tumor development, even though the compound does not have antiviral activity for HBV or HCV by itself. Therefore, suppression of hepatic necroinflammation by these drugs may serve to prevent hepatocarcinogenesis. In contrast, acyclic retinoid suppresses the post‐therapeutic recurrence in cirrhotic patients who underwent curative treatment of preceding tumors. The retinoid induces the disappearance of serum lectin‐reactive α‐fetoprotein (AFP‐L3), a tumor marker indicating the presence of unrecognizable tumors in the remnant liver, suggesting a deletion of such minute (pre)malignant clones (clonal deletion). As a molecular mechanism of the clonal deletion, a novel mechanism of apoptosis induction by the retinoid via tissue transglutaminase is implicated. In future, a combination of immunopreventive and chemopreventive therapies may give a clue to the further advances of cancer prevention, and thereby to the improvement of the prognosis of cirrhotic patients.

Autonomic nervous function in upper gastrointestinal endoscopy : a prospective randomized comparison between transnasal and oral procedures

Background. Transnasal esophagogastroduodenoscopy (EGD) using an ultrathin endoscope is less stressful to the cardiovascular system with less elevation of systolic blood pressure (BP) than oral procedures. To elucidate the mechanism of such beneficial cardiovascular responses, we performed a prospective patient-centered randomized study in which BP and pulse rate (P), as well as autonomic nervous functions, were estimated during transnasal EGD compared with those in oral procedures using the same ultrathin endoscope. Methods. The study involved 781 patients, among whom 55 and 56 cases were assigned to transnasal and oral EGD groups, respectively. The autonomic nervous responses were determined employing power spectral analysis (PSA) of heart-rate variations on electrocardiogram. PSA data were based on two peaks in lowfrequency (LF) and high-frequency (HF) ranges. HF power and the ratio of LF power/HF power represented parasympathetic and sympathetic nervous activities, respectively. Results. Our study confirmed the lesser elevation of BP and P in patients undergoing transnasal EGD than in those undergoing oral procedures. PSA revealed a lower increase in LH power/HF power in transnasal EGD than in oral EGD. However, both endoscopic procedures equally suppressed HF power. Significant correlations were found between the parameters of cardiovascular response (P and BP) and autonomic functions (LF power/HF power ratio and HF power). Conclusions. This is the first study demonstrating less sympathetic stimulation in patients undergoing transnasal EGD, leading to lesser elevation of BP and P.

Reduction of serum lipoprotein(a) levels in hyperlipidaemic patients with α-tocopheryl nicotinate

Abstract The effect of low dose (600 mg/day) α-tocopheryl nicotinate on serum lipoprotein(a) (Lp(a)) concentration was studied in 28 hyperlipidaemic patients. Serum lipids, lipoproteins and apolipoproteins, except for Lp(a), tended to increase after treatment. In particular, the changes in HDL-cholesterol and apo C-II levels were statistically significant. On the other hand, serum Lp(a) levels in all patients decreased significantly after 2 months of treatment. Furthermore, no difference between before and after treatment was observed in the group with initial Lp(a) levels