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Abstract: Background/Aim: Ubiquitin (Ub)-dependent degradation of regulatory proteins controls many cellular processes such as cell cycle progression, morphogenesis and signal transduction. In this study, we evaluated the meaning of ubiquitination in chronic liver diseases, especially human hepatocellular carcinoma, with regard to recurrence.
Methods: A total 74 of liver tissues (8 of chronic hepatitis [CH], 9 of liver cirrhosis [LC], 7 of dysplastic nodule low grade (DSL), or dysplastic nodule high grade (DSH) and 50 of hepatocellular carcinoma [HCC]) were analyzed for ubiquitination by immunohistochemisty. Cell proliferation was also analyzed using Ki-67 staining. As a comparative marker for progression of HCC, PIVKA-II (protein induced by vitamin K absence-II) was employed to examine the recurrence rate of HCC.
Results: Ubiquitin (Ub) was positive in nuclei and cytoplasm of HCC in immunohistochemistry. The labeling index (L.I.) of ubiquitination was significantly higher with HCC than with other chronic liver diseases and tended to correlate with the lack of poorly- differentiated of HCC. The L.I. of Ki-67 staining was also correlated (P < 0.0001) with that of ubiquitination. The hepatocellular carcinoma (HCC) samples from potentially curatively operated patients having a ubiquitination L.I. of more than 20% suffered significantly higher recurrence of HCC than did patients with an L.I. of less than 20%. On the other hand, PIVA-II did not show such a difference.
Conclusion: Ubiquitin (Ub) may reflect the growth activity of neoplasms and will be a possible new predictive marker for the recurrence of human hepatocellular carcinoma after potentially curative operation.
The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin (Ub)-mediated pathway. The elucidation of this system began in 1978, when Hershko et al. characterized a heat-stable polypeptide required for the activity of an ATP-dependent proteolytic system from reticulocytes (1). This polypeptide was subsequently identified as ubiquitin (Ub), a 76 amino acid residue, highly conserved protein present in all eukaryotes. In 1981, it was reported that Ub is covalently ligated to protein substrates in an ATP-dependent reaction (2) and it was proposed that Ub ligation commits proteins for degradation (3–5). The ubiquitination of proteins has been implicated in a variety of processes such as the heat shock response, DNA repair, cell cycle progression, the modification of histones and of receptors, and the possible pathogenesis of some neurodegenererative diseases. The pathways of ubiquitination of proteins have been studied (6–12). Ubiquitin (Ub) covalently attaches to various target proteins with the help of Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), and the recognizing protein, Ub-ligated protein (E3) (5,13). Recently, the accumulation of Ub or Ub-conjugated proteins has been observed immunohistochemically in many human malignant tumors, including astrocytoma, lung cancer, prostate cancer, and hepatocellular carcinoma (HCC) (14–17). It was reported that the intensity of Ub staining appeared to increase in advanced HCC; however, in these advanced HCC cases no specific correlation was found between Ub immunoreactivity and differentiation of HCC (18). The target proteins for ubiquitination and the meaning of ubiquitination have not yet been well evaluated in liver disease.
Protein induced by vitamin K absence-II (PIVKA-II) has been considered to be a useful serum marker of HCC. Liebman et al. first described it as a tumor marker for HCC in 1984 (19). Recently, Tamano et al. reported that the PIVKA-II positive rate of the tumors was 81.8%, and that PIVKA-II staining tended to be more intense in tissues from patients with portal tumor thrombus, distant metastasis, or a longer duration of disease history of HCC (20).
In the present study, we analyzed ubiquitination in samples of HCC and chronic liver diseases and evaluate the meaning of Ub staining for the prognosis of HCC by comparing it with PIVKA-II.
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- Materials and methods
Several reports have described the increased intensity of ubiquitin (Ub) immunoreactivity in neoplastic cells (14–17), which suggests the accumulation of free Ub or ubiquitinated proteins. One possible explanation for this accumulation is that these accumulated proteins may be specific proteins of neoplastic cells independent of Ub-dependent proteolysis. Indeed, high expression of Ub and proteosomes at the mRNA level in renal cancer has been reported (15).
Although we cannot exclude this, there is also a possibility that Ub will play an important role as a degradation marker. The central mechanism in the degradation of proteins in the cell is the protein resolution (degradation) system dependent on the energy of Ub and proteosomes. The cyclin-dependent kinase inhibitor (CKI) is regulated by the Ub system. P27 is known as an inhibitor of CKI and there are increasing reports of a decrease of P27 expression and the appearance of carcinoma (22–24). Thus, Ub seems to play critical roles in various important physiological phenomena, e.g. the cell cycle and carcinogenesis.
In this manuscript, we have shown that Ub was mainly detected in the nuclei and cytoplasm of HCC cells; however, PIVKA-II was mainly detected in the cytoplasm of HCC cells. There are many reports that various proteins such as cell cycle regulators are ubiquitinated (25,26). Therefore, it is reasonable that Ub was detected both in the nuclei and cytoplasm in HCC cells, and it is speculated that many proteins such as cell cycle regulators and transcription factors will be ubiquitinated. Previously, Osada et al. (18) reported the possible relationship between the appearance of ubiquitinated proteins and multi step hepatocarcinogenesis, but they did not examine the relationship with Ki-67 or prognosis.
Ki-67 has been reported to be a good proliferation marker for various cells (27). In this study, Ub also seemed to be a good proliferation marker for HCC, because the L.I. of Ub correlated with that of Ki-67.
PIVKA-II is also a well-confirmed useful marker for HCC progression (20). Thus, we tried to evaluate the meaning of Ub staining compared with PIVKA-II. We analyzed Ub staining especially with regard to the recurrence rate of HCC.
The ubiquitin L.I. of HCC was significantly higher than that of chronic liver diseases and tended to increase in proportion to histological poorly-differentiated of HCC. On the other hand, the L.I. of PIVKA-II seemed to be increased at the stage of dysplastic nodule low grade (DSL), or dysplastic nodule high grade (DSH), but there was no correlation with histological poorly- differentiated of HCC.
The operated patients whose L.I. of Ub in HCC samples were more than 20% clearly showed a higher incidence of tumor recurrence. These results demonstrate that Ub is a better predictive marker of the prognosis of HCC than PIVKA-II after operation. We have confirmed the result reported by Kuang-Liang King et al. (28) showing that potentially curatively operated patients with HCC having an L.I. of Ki-67 of more than 10% had a significantly (P < 0.05) lower tumor-free survival rate than those with one of less than 10% (data not shown). However, there has been no report concerning the ubiquitin L.I. and tumor recurrence so far. The conclusion of the present study is that ubiquitin is a potentially better candidate as a predictive marker for the recurrence of human hepatocellular carcinoma than PIVKA-II.