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Keywords:

  • hepatocellular carcinoma – PIVKA-II – recurrence – tumor marker – ubiquitin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tissue specimens

A total of 74 samples (from 56 males and 18 females) were obtained for this study, including eight from chronic hepatitis (CH), nine from liver cirrhosis (LC), seven from dysplastic nodules, low grade (DSL) or high grade (DSH) and 50 from HCC (17 well, 22 moderately, and 11 poorly differentiated). All of the HCC samples were surgically resected in potentially curative operations at the Yamaguchi University Hospital. All samples of CH and LC and DSL/DSH were obtained by ultrasonography-guided biopsy at the Yamaguchi University Hospital. They were histologically confirmed as CH, LC or DSL/DSH (Table 1).

Table 1.  Patient details
 CHLCDSL/DSHWellModeratePoorTotal
M/F4/4 (8)7/2 (9)5/2 (7)14/3 (17)18/4 (22)8/3 (11)56/18
B/C/non B C3/5/03/5/11/5/13/11/35/16/13/8/018/50/6
Age45.0 ± 14.146.7 ± 14.163.9 ± 4.362.5 ± 7.463.1 ± 10.559.0 ± 12.0 

Tissues were fixed in 10% buffered formalin and embedded in paraffin. The specimens were cut into 3 micron sections and placed on glass slides for immunohistochemical staining. Immunohistochemical staining was performed using a DAKO LSAB KIT (DAKO JAPAN, Kyoto, Japan) as described elsewhere. MU-3 antibody (a kind gift from Eisai Pharmaceutical Company, LTD, Tokyo, Japan) was used as the PIVKA-II antibody. Ubiquitin antibody (Sigma-Aldrich.com) and MIB-1 antibody as the Ki-67 antibody (MBL, IM0505) were also used. The percentages of Ki-67, PIVKAII and ubiquitin-positive cells in 1000 tumor cells were calculated as the labeling indices (L.I.). Among 50 HCC cases, three cases for ubiquitin, three cases for Ki-67 and two cases for PIVKA II staining were completely negative and regarded as labeling index.

Statistical analysis

Recurrence-rate analysis was performed for HCC patients using the Kaplan-Meier product-limit method (21). Differences between groups were tested using the T-proficiency test. The relationship of tissue PIVKA-II staining and tissue Ki-67 staining in HCC was tested by the correlation of Pearson's analysis.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Immunohistochemical staining for ubiquitin

Ubiquitin (Ub) was detected in the nuclei and cytoplasm of HCC cells. The intensity of Ub staining is shown as a labeling index determined by counting the number of positive cells per 1000 tumor cells (Fig. 1A).

image

Figure 1. Immunohistochemical staining of ubiquitin in HCC.(A) Ubiquitin immunoreactivity was observed in tumor cells but not in non-tumor cells. Coarse granular staining is seen in both the cytoplasm and nucleus. (X 400) (B) Ki-67 expression in tumor cells. Coarse granular staining is seen only in nuclei. (X 400) (C) PIVKA-II expression in tumor cells. Coarse granular staining is seen in the cytoplasm but not in the nucleus. (X 400)

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The Ub L.I. of chronic liver diseases was as follows: CH, 33.8 + 25.6 (n = 8); LC, 28.9 + 41.4 (n = 9); and DSL/DSH, 52.9 + 70.4 (n = 7).

On the other hand, HCC had a significantly higher L.I. (Fig. 2A). For well-differentiated HCC it was 238.4 + 178.7 (n = 16), for moderately differentiated HCC, 296.4 + 217.7 (n = 23), and for poorly differentiated HCC 358.9 + 279.7 (n = 11). The L.I. of Ub tended to increase in proportion to the extent of undifferentiation.

image

Figure 2. Immunohistochemical staining and histological differentiation of HCC.(A) Ubiquitin (Ub) staining and histological differentiation of HCC. Ub staining is stronger in HCC than in non-tumor cells (P < 0.05), but no significant difference was noted between the degrees differentiation of HCC.(B) PIVKA-II staining and the histological differentiation of HCC. PIVKA-II staining tended to be stronger in HCC than in non-tumor cells (P < 0.05), but no significant difference was seen between the degrees of differentiation of HCC.

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Ubiquitin and Ki-67

MIB-1 (Ki-67) was mainly detected in the nuclei of HCC cells. The intensity of Ki-67 staining is also shown as a labeling index representing the number of Ki-67-positive cells per 1000 tumor cells (Fig. 1B). The relationship of Ub and Ki-67 staining of HCC is shown in Fig. 3A. The L.I. of ubiquitin correlated with that of Ki-67. (r = 0.520, P < 0.00001).

image

Figure 3. Relationship between ubiquitin (Ub) and PIVKA-II and Ki-67(A) The relationship of Ub and Ki-67 staining in HCC. The labeling index (L.I.) of Ub correlated with that of Ki-67 (r = 0.520, P < 0.00001). The y-axis is the Ki-67 L.I, and the x-axis is the ubiquitin L.I. (B) The relationship of the L.I. of PIVKA-II and Ki-67 in HCC. The L.I. of PIVKA-II correlated with that of Ki-67 (r = 0.398, P < 0.0038). The y-axis is L.I. of Ki-67, and the x-axis is that of PIVKA-II.

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Ubiquitin L.I. and recurrence rate of HCC

Long-term observation after potentially curative-operation was achieved for 38 of 50 patients. The L.I. of ubiquitination in chronic liver disease was less than 20%. Thus the L.I. of HCC was divided into three groups (less than 20% [A], 20%−40% [B], and more than 40% [C]). The distributions of age and sex were not significantly different among these groups. An increased L.I. of Ub clearly indicated a significantly higher recurrence rate of HCC after potentially curative operation (Fig. 4A). Thus, Ub seemed to be a good predictive marker of recurrence of HCC after operation.

image

Figure 4. The relationship of immunohistochemical staining and tumor-free survival after potentially curative operation or HCC.(A) Ub staining tended to be stronger in cases with early recurrence. The labeling index (L.I.) of immunohistochemical staining was divided into the three groups (L.I. < 20% (i), 20% < L.I. > 40% (ii), and L.I. > 40% (iii)).The patients whose resected HCC samples had a higher L.I. of Ub clearly had more recurrence of HCC compared to the patients with a lower L.I. of Ub. The L.I. of PIVKA-II was divided into three groups just as with ubiquitin staining: (L.I. < 20% (i), 20% < L.I. > 40% (ii), and L.I < 40% (iii)). There was a significant difference only between group A and group C.

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PIVKA-II staining

PIVKA-II was mainly detected in the cytoplasm of HCC cells. The intensity of PIVKA-II staining is shown as a labeling index as for Ub and Ki-67 (Fig. 1C). The L.I. of PIVKA-II of chronic liver diseases was as follows: CH, 47.5 + 54.7 (n = 8); LC, 35.6 + 43.3 (n = 9); and DSL/DSH, 130.0 + 172.4 (n = 7).

On the other hand, for well-differentiated HCC it was 193.6 + 195.0 (n = 16), for moderately differentiated HCC 317.4 + 198.6 (n = 23), and for poorly differentiated HCC 298.1 + 230.9 (n = 11). These results indicated that PIVKA-II tended to increase already at the stage of DSL/DSH but no significant correlation was found between tissue PIVKA-II staining and the histological differentiation of HCC (Fig. 2B).

Relationship between PIVKA-II and Ki-67

The relationship of the PIVKA-II and Ki-67 lI is shown in Fig. 3B. There was a weak correlation between the L.I. of PIVKA-II and Ki-67 (r = 0.398, P = 0.0038.).

PIVKA-II and recurrence rate of HCC

The recurrence rate of HCC was examined with the same patients studied for the L.I. of Ub. As the L.I. of PIVKA-II was also less than 20% in chronic liver disease, the L.I. was divided into three groups (less than 20% [A], 20% to 40% [B], and more than 40% [C]) Distributions of age or sex were not significantly different among these groups. However, there was a significant difference (P < 0.05) between groups A and C with respect to survival (Fig. 4B).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported in part by Grants-in-Aid 11670507 and 13470121 from the Ministry of Education, Science and Culture, Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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