Japanese Journal of Clinical Oncology 31:359-362 (2001)
© 2001 Foundation for Promotion of Cancer Research
Apoptosis Induction by Acyclic Retinoid: a Molecular Basis of ‘Clonal Deletion’ Therapy for Hepatocellular Carcinoma
1First Department of Internal Medicine and 2Department of Molecular Pathobiochemistry, Gifu University School of Medicine, Gifu and 3Laboratory of Molecular Cell Sciences, Tsukuba Institute, The RIKEN, Tsukuba, Ibaraki, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT CLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
We have shown previously that administration of acyclic retinoid to cirrhotic patients who had undergone curative treatment of preceding hepatocellular carcinoma (HCC) induced the disappearance of serum lectin-reactive
-fetoprotein (AFP-L3) and subsequently reduced the incidence of second liver cancers. AFP-L3 is a tumor marker that indicates the presence of occult tumors below the detection limit by diagnostic images. Therefore, we have proposed a new concept of ‘clonal deletion’ therapy with acyclic retinoid for the cancer chemoprevention against HCC. Such eradication of AFP-L3-producing latent malignant (or premalignant) cells from the liver suggested a new strategy to prevent HCC, which may be involved in the same category as cancer chemotherapy. In the present series of studies, we explored the molecular mechanism of ‘clonal deletion’ and found a novel mechanism of apoptosis induction by the retinoid. We have demonstrated a modification of a retinoid receptor, RXR, by mitogen-activated protein (MAP) kinase-dependent phosphorylation, resulting in the loss of transactivating activity. This may lead HCC cells to be resistant to natural retinoic acid. However, acyclic retinoid restored the function of phosphorylated RXR and induced its downstream pro-apoptotic genes including tissue transglutaminase, an enzyme that is implicated in apoptosis. Tissue transglutaminase-dependent apoptosis in HCC cells was independent of the activation of caspases. This novel mechanism of retinoid-induced apoptosis may give a clue to understand the molecular mechanism of clonal deletion. |
CLINICAL BACKGROUND |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
Hepatocellular carcinoma (HCC) is a major health problem throughout the world, including the USA (1), Western Europe (2,3) and Eastern Asia (4). The incidence of HCC has been increasing recently and thus attention has been focused on the disease in these countries. The annual incidence of HCC reaches 5–8% both in type B and C hepatitis virus-infected cirrhotic patients (5). Moreover, the incidence leaps to ~20% a year in cirrhotic patients who have undergone curative treatment of the primary HCC. This high incidence of second HCC, half of which is the second primary cancer and is not an intrahepatic metastatic tumor, is a major cause of a low 5-year survival rate (40%) after the curative treatment of HCC (6). Therefore, future advances only in medical techniques for both the early detection and therapy of HCC may be insufficient for the further improvement of the therapeutic outcome of HCC, and a new strategy to prevent second primary HCC may be required for this purpose. Therefore, chemoprevention of hepatocellular carcinoma (HCC) is of great significance because of the high incidence of the latter in viral-associated cirrhotic patients.
Some strategies have been reported for suppressing hepatocarcinogenesis in clinical trials, including interferon and ß (7), a Japanese herbal medicine, TJ-9 (8), glycyrrhizin (9) and our retinoid analog, acyclic retinoid (10,11). Interferon, TJ-9 and glycyrrhizin may belong to a category of immunopreventive agents, whereas acyclic retinoid is a member of chemopreventive agents. Interferon, TJ-9 and glycyrrhizin have been shown to prevent successfully the occurrence of primary HCC in cirrhotic patients and acyclic retinoid suppressed the appearance of second HCC after curative treatment of preceding tumors.
|
RETINOID AND ‘CLONAL DELETION’ |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
Acyclic retinoid (all-trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentanoic acid) or NIK 333 (Nikken Chemicals, Tokyo, Japan) inhibited experimental liver carcinogenesis (12) and induced the apoptosis of human hepatoma-derived cell lines (13). In an experimental rat model treated with 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB), acyclic retinoid suppressed the appearance of oval cells, progenitor cells of HCC and cholangiocellular carcinoma (CCC), in the early stage of carcinogenesis and the subsequent development of both HCC and CCC in the late stage (T. Tanaka, T. Sano, et al., unpublished observations). In addition, acyclic retinoid suppressed dimethylnitrosamine (DEN)-induced HCC in rats, when administered in the promotion phase (T. Tanaka, T. Sano, et al., unpublished observations). Thus, the retinoid seemed to exert preventive effects on hepatocarcinogenesis in both early and late phases.
We further examined the chemopreventive effect of acyclic retinoid on HCC in a clinical intervention study. We have demonstrated that oral administration of acyclic retinoid for 12 months significantly reduced the incidence of second primary cancers in patients who had received curative treatment of HCC in a randomized controlled trial (10). Moreover, the survival rate was also significantly improved by the compound after a median observation period of 62 months in the follow-up study (11). In that clinical trial, serum lectin-reactive -fetoprotein (AFP-L3), which indicates the presence of immature hepatic cells in the remnant liver, disappeared in the acyclic retinoid group after 12 months of administration. This observation suggests that AFP-L3-producing clones were eradicated by acyclic retinoid from the remnant liver (14). In contrast, a spontaneous reduction in serum AFP-L3 levels was not observed in the placebo group. Therefore, we suggested that acyclic retinoid deleted premalignant or latent malignant clones that produce AFP-L3 from the remnant liver. Moreover, acyclic retinoid suppressed the appearance of serum AFP-L3 in patients whose AFP-L3 levels were below the detection range at entry, whereas the number of patients whose serum AFP-L3 newly appeared was significantly increased in the placebo group. From these results, we proposed the new concept of cancer chemoprevention, ‘clonal deletion’ and ‘clonal inhibition’ (Fig. 1) (15). Because of a high incidence of HCC in cirrhotic patients, it may well be possible that there exist some latent malignant (or premalignant) cells in the cirrhotic liver that cannot be detected by diagnostic images. Acyclic retinoid might remove such clones from the liver and thereby contribute to reducing the incidence of second HCC. These ideas may allow us to place chemoprevention in the same category as chemotherapy.
|
|
RETINOID RESISTANCE IN HCC |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
We have shown a depletion of retinoid in tumorous tissues in both experimental and clinical HCCs (12). The absence of hepatic stellate cells, retinoid-storing cells in the liver, is a major cause of such retinoid depletion in the tumors. In addition, a rapid metabolism of retinoid into an inactive metabolite may well participate in such depletion (12). In fact, retinol has been shown to be rapidly converted to anhydroretinol, an inactive retinol metabolite that cannot be metabolized to retinoic acid, a ligand of retinoid nuclear receptor. This retinoid depletion might be involved in hepatocarcinogenesis. For instance, we have suggested that the enhanced loss of retinoid in the liver in cirrhotics with viral infection plus alcohol abuse may be linked to the accelerated incidence of HCC (16). Since there have been several reports showing chemopreventive effects of retinoids on liver cancer in both experimental and clinical studies, it seems conceivable that retinoid deficiency may promote tumorigenesis in the liver.
In addition to retinoid depletion, we have also found that a malfunction of retinoid nuclear receptor in hepatoma cells may contribute to hepatocarcinogenesis. Retinoids exert their biological functions through two distinct nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR) (17). RAR interacts with both all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9cRA), whereas RXR binds only to 9cRA and regulates the expression of several target genes. Both RAR and RXR consist of three subtypes, , ß and 
, characterized by a modular domain structure. RXR forms a homodimer in addition to heterodimers with RAR and other nuclear receptors. These dimers bind to their respective response elements and subsequently activate or inhibit the expression of their target genes. RAR and RXR bind to a retinoic acid response element (RARE) and retinoid X response element (RXRE), respectively. It is suggested that both RAR and RXR are involved in hepatocarcinogenesis. For example, expression of RAR gene is localized near the integration site of hepatitis B virus and is induced in HCC tissue (18). On the other hand, RXR is reported to bind to the enhancer element of hepatitis B virus and modulate viral replication (19).
Among these receptors, RXR is most abundant in the liver and highly expressed in HCC cells. We have shown a delay in the metabolism of RXR that stayed in a full-length 54 kDa protein, whereas RXR rapidly decayed into smaller peptides in the surrounding cirrhotic and normal liver tissues (20). Because there was no genomic mutation in RXR gene in either surgically resected human HCC tissues or human HCC cell lines (S. Adachi, et al., unpublished observations), we hypothesized that post-translational modification of RXR may take place and be responsible for the delayed proteolytic decay of the receptor. Many members of the nuclear hormone receptor family are phosphoproteins (21). Recently, phosphorylation of RXR by mitogen-activated protein (MAP) kinase has been reported in keratinocytes, which may play an important role in their malignant transformation (22). We have reported that the non-lysosomal calcium-dependent cysteine protease m-calpain participates in the proteolysis of RXR (23). Some substrates of m-calpain, such as c-Fos, may become resistant to proteolysis after their phosphorylation (24). Very recently, we have found that full-length RXR protein was phosphorylated, but this was not the case with proteolytically truncated RXR protein, in both surgically resected tissues and HCC cell lines (R. Matsushima-Nishiwaki, et al., unpublished observations) (Fig. 2). Phosphorylation took place at some serine and threonine residues, consensus sites of MAP kinases that played a significant role in such phosphorylation. Interestingly, phosphorylated RXR lost its transactivating activity and this seems to correlate with enhanced proliferation of HCC cells. Although we have not yet fully identified the target gene(s) of RAR that regulate cellular proliferation, these observations may suggest that malfunction of phosphorylated RXR is linked to the aberrant growth of HCC. Therefore, in HCC tissues and cells, not only retinoid depletion but also malfunction of modified retinoid nuclear receptor may be involved in the development of HCC.
|
|
APOPTOSIS BY ACYCLIC RETINOID |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
‘Clonal deletion’ therapy suggests a removal of latent malignant (or premalignant) cells from the liver. In general, two possible mechanisms are widely accepted for such removal of transformed cells: cell death (or apoptosis) and differentiation induction. These two mechanisms may work independently in some cases, but coincide in many systems. A typical example is retinoic acid therapy against acute promyelocytic leukemia (APL) (25). Retinoic acid induces the differentiation of APL cells into granulocytes. Once differentiated, granulocytes restore their normal function and terminate their lives by apoptosis. This phenomenon is recognized as terminal differentiation. Similar mechanisms are hypothesized also in the chemoprevention of several cancers.
In the case of HCC, we have shown the induction of both apoptosis (13) and differentiation induction (26). At present, differentiation of HCC cells by acyclic retinoid still requires further investigation. We only have shown the up-regulation of albumin and down-regulation of -fetoprotein, genes characteristic of differentiated and undifferentiated hepatocytes, respectively (26), but failed to show the phenotypic changes of HCC cells. In contrast, apoptosis induction of HCC cells by acyclic retinoid has been shown clearly and its molecular mechanism has been elucidated. Here, immediate apoptosis that is not accompanied by differentiation induction has been studied. The involvement of tissue transglutaminase in the retinoic acid-induced apoptosis in APL cells has been proposed, which may take place independently of the activation of caspases (S. Kojima, unpublished observation). There have been several reports showing an induction of tissue transglutaminase in retinoic acid-induced apoptotic cells (27). However, the function of tissue transglutaminase is not yet understood. We found that acyclic retinoid-induced apoptosis in the HCC cell line HuH7 was also dependent on tissue transglutaminase (T. Sano, unpublished observations). This apoptosis was not inhibited by caspase inhibitors, but was suppressed by antisense oligo DNA specific to tissue transglutaminase. However, the HCC cells treated with acyclic retinoid showed typical features of apoptotic changes including both chromatin condensation when examined by electron microscopy and DNA ladder formation. We therefore speculate that both tissue transglutaminase and caspase work simultaneously; the former might participate in the chromatin condensation, whereas the latter works for DNA fragmentation.
Tissue transglutaminase is one of the downstream genes regulated by the RAR/RXR system. Thus, it might well be possible that malfunction of phosphorylated RXR results in the failure of tissue transglutaminase induction in HCC cells, leading to the uncontrolled proliferation that is resistant to natural retinoic acid. However, acyclic retinoid might somehow restore the function of RXR and thereby induce apoptosis via the tissue transglutaminase pathway. The mechanism by which acyclic retinoid recovers phosphorylated RXR function is now under investigation.
|
CLINICAL APPLICATION OF CLONAL DELETION |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
‘Clonal deletion’ therapy has already been theoretically proposed as a mechanism of cancer chemoprevention (28–30) and was strongly supported by our clinical experience of HCC (15). The present study may provide information to understand the molecular mechanism of ‘clonal deletion’. The benefit of clinical application of this new concept may place chemoprevention in the same category as chemotherapy, which will make prevention more easily acceptable in the clinical field. It is widely recognized that there may be minute or latent malignant cells in the cirrhotic liver in patients such as AFP-L3-positive. Therefore, even when the cells cannot be detected by diagnostic images, there seem to be good reasons to treat such patients with ‘clonal deletion’ therapy.
|
Acknowledgment |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
This work was supported in part by the Foundation for Promotion of Cancer Research in Japan (to H.M.).
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Hisataka Moriwaki, First Department of Internal Medicine, Gifu University School of Medicine, Gifu 500-8705, Japan
Abbreviations: AFP-L3, lectin-reactive -fetoprotein; APL, acute promyelocytic leukemia; CCC, cholangiocellular carcinoma; DEN, dimethylnitrosamine; HCC, hepatocellular carcinoma; MAP, mitogen-activated protein; 3'MeDAB, 3'-methyl-4-dimethylaminoazobenzene; RA, retinoic acid; RXR, retinoid X receptor.
|
REFERENCES |
|---|
|
TOPABSTRACTCLINICAL BACKGROUNDRETINOID AND ‘CLONAL...RETINOID RESISTANCE IN HCCAPOPTOSIS BY ACYCLIC RETINOIDCLINICAL APPLICATION OF CLONAL...AcknowledgmentREFERENCES |
|---|
1 El-Serag HB, Manson AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 2000;340:745–50.
2 Taylor-Robinson SD, Foster GR, Arora S, Hargreaves S, Thomas HC. Increase in primary liver cancer in the UK, 1979–94. Lancet 1997;350:1142–3.[Medline]
3 Deuffic S, Poynard T, Buffat L, Valleron A-J. Trends in primary liver cancer. Lancet 1998;351:214–5.[Web of Science][Medline]
4 Tobe T, Kameda H, Okudaira M, Ohto M, Endo Y, Mito M, et al., editors. Primary Liver Cancer in Japan. Springer: Tokyo 1992.
5 Shiratori Y, Yoshida H, Omata M. Different clinicopathological features of hepatocellular carcinoma in relation to causative agents. J Gastroenterol 2001;36:73–8.[Web of Science][Medline]
6 Liver Cancer Study Group of Japan. Survey and follow-up study of primary liver cancer in Japan. Kanzo 1997;38:317–30 (in Japanese).
7 Nishiguchi S, Kuroki T, Nakatani S, Morimoto H, Taketa T, Nakajima S, et al. Randomized trial of effects of interferon-alpha on the incidence of hepatocellular carcinoma in chronic active hepatitis C with cirrhosis. Lancet 1995;346:1051–5.[Web of Science][Medline]
8 Oka H, Yamamoto S, Kuroki T, Harihara S, Marumo T, Kim SR, et al. Prospective study of chemoprevention of hepatocellular carcinoma with Sho-saiko-to (TJ-9). Cancer 1995;76:743–9.[Web of Science][Medline]
9 Arase Y, Ikeda K, Murashima N, Chayama K, Tsubota A, Koida I, et al. The long term efficacy of glycyrrhizin in chronic hepatitis C patients. Cancer 1997;79:1494–500.[Web of Science][Medline]
10 Muto Y, Moriwaki H, Ninomiya M, Adachi S, Saito A, Takasaki KT, et al. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. N Engl J Med 1996;334:1561–7.
11 Muto Y, Moriwaki H, Saito A. Prevention of second primary tumors by an acyclic retinoid in patients with hepatocellular carcinoma. N Engl J Med 1999;340:1046–7.
12 Muto Y, Moriwaki H. Antitumor activities of vitamin A and its derivatives. J Natl Cancer Inst 1984;73:1389–93.
13 Nakamura N, Shidoji Y, Yamada Y, Hatakeyama H, Moriwaki H, Muto Y. Induction of apoptosis by acyclic retinoid in the human hepatoma-derived cell line, HuH-7. Biochem Biophys Res Commun 1995;207:382–8.[Web of Science][Medline]
14 Moriwaki H, Yasuda I, Shiratori Y, Uematsu T, Okuno M, Muto Y. Deletion of serum lectin-reactive -fetoprotein by acyclic retinoid: a potent biomarker in the chemoprevention of second primary hepatoma. Clin Cancer Res 1997;3:727–31.[Abstract]
15 Moriwaki H, Okuno M, Shiratori Y, Yasuda I, Muto Y. Clonal deletion, a novel strategy of cancer control that falls between cancer chemoprevention and cancer chemotherapy: a clinical experience in liver cancer. In: Okita K, editor. Frontiers in Hepatology. Progress in Hepatocellular Carcinoma Treatment. Springer: Tokyo 2000;97–103.
16 Adachi S, Moriwaki H, Muto Y, Yamada Y, Fukutomi Y, Shimazaki M, et al. Reduced retinoid content in hepatocellular carcinoma with special reference to alcohol consumption. Hepatology 1991;14:776–80.[Web of Science][Medline]
17 Mangelsdorf DJ, Umesono K, Evans RM. The retinoids receptors. In: Roberts AB, Goodman DS, Sporn MB, editors. The Retinoids: Biology, Chemistry and Medicine, 2nd ed. Raven Press: New York 1994;319–49.
18 Benbrook D, Lernhardt E, Pfahl M. A new retinoic acid receptor identified from a hepatocellular carcinoma. Nature 1988;333:669–72.[Medline]
19 Garcia AD, Ostapchuk P, Hearing P. Functional interaction of nuclear factors EF-C, HNF-4 and RXR with hepatitis B virus enhancer 1. J Virol 1993;67:3940–50.
20 Matsushima-Nishiwaki R, Shidoji Y, Moriwaki H, Muto Y. Aberrant metabolism of retinoid X receptor proteins in human hepatocellular carcinoma. Mol Cell Endocrinol 1996;121:179–90.[Web of Science][Medline]
21 Weigel N. Steroid hormone receptors and their regulation by phosphorylation. Biochem J 1996;319:657–67.
22 Solomon C, White JH, Kremer R. Mitogen-activated protein kinase inhibits 1,25-dihydroxyvitamin D3-dependent signal transduction by phosphorylating human retinoid X receptor . J Clin Invest 1999;103:1729–35.[Web of Science][Medline]
23 Matsushima-Nishiwaki R, Shidoji Y, Nishiwaki S, Moriwaki H, Muto Y. Limited degradation of retinoid X receptor by calpain. Biochem Biophys Res Commun 1996;225:946–51.[Web of Science][Medline]
24 Okazaki K, Sagata N. The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH3T3 cells. EMBO J 1995;14:5048–59.[Web of Science][Medline]
25 Huang ME, Yu-Chen Y, Shu-Rong C, Lu MX, Zhao L, Gu LJ, et al. Use of all-trans-retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988;72:567–72.
26 Yamada Y, Shidoji Y, Fukutomi Y, Ishikawa T, Kaneko T, Nakagama H. Positive and negative regulations of albumin gene expression by retinoids in human hepatoma cell lines. Mol Carcinog 1994;10:151–8.[Web of Science][Medline]
27 Nagy L, Thomazy VA, Heyman RA, Davies PJA. Retinoid-induced apoptosis in normal and neoplastic tissues. Cell Death Different 1998;5:11–9.[Web of Science][Medline]
28 Hong WK, Lippman SM, Hittelman WN, Lotan R. Retinoid chemoprevention of aerodigestive cancer: from basic research to the clinic. Clin Cancer Res 1995;1:677–86.[Abstract]
29 Lotan R. Retinoids in cancer in cancer chemoprevention. FASEB J 1996;10:1031–9.[Abstract]
30 Lotan R. Retinoids and apoptosis: implications for cancer chemoprevention and therapy. J Natl Cancer Inst 1995;22:1655–7.
Received February 28, 2001; accepted April 9, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Matsushima-Nishiwaki, M. Okuno, Y. Takano, S. Kojima, S. L. Friedman, and H. Moriwaki Molecular mechanism for growth suppression of human hepatocellular carcinoma cells by acyclic retinoid Carcinogenesis, August 1, 2003; 24(8): 1353 - 1359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Okada Cancer Chemoprevention as Adjuvant Therapy for Hepatocellular Carcinoma Jpn. J. Clin. Oncol., August 1, 2001; 31(8): 357 - 358. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||