Original Contribution
Inflammation-induced protein carbonylation contributes to poor prognosis for cholangiocarcinoma

https://doi.org/10.1016/j.freeradbiomed.2012.01.018Get rights and content

Abstract

Carbonylation is an irreversible and irreparable protein modification induced by oxidative stress. Cholangiocarcinoma (CCA) is associated with chronic inflammation caused by liver fluke infection. To investigate the relationship between protein carbonylation and CCA progression, carbonylated proteins were detected by 2D OxyBlot and identified by MALDI-TOF/TOF analyses in pooled CCA tissues in comparison to adjacent nontumor tissues and normal liver tissues. We identified 14 highly carbonylated proteins in CCA tissues. Immunoprecipitation and Western blot analyses of individual samples confirmed significantly greater carbonylation of serotransferrin, heat shock protein 70-kDa protein 1 (HSP70.1), and α1-antitrypsin (A1AT) in tumor tissues compared to normal tissues. The oxidative modification of these proteins was significantly associated with poor prognoses as determined by the Kaplan–Meier method. LC–MALDI-TOF/TOF mass spectrometry identified R50, K327, and P357 as carbonylated sites in serotransferrin, HSP70.1, and A1AT, respectively. Moreover, iron accumulation was significantly higher in CCA tissues with, compared to those without, carbonylated serotransferrin. We conclude that carbonylated serotransferrin-associated iron accumulation may induce oxidative stress via the Fenton reaction, and the carbonylation of HSP70.1 with antioxidative property and A1AT with protease inhibitory capacity may cause them to become dysfunctional, leading to CCA progression.

Graphical abstract

Highlights

► In liver fluke-associated cholangiocarcinoma tissues, inflammation resulted in carbonylated proteins. ► Carbonylation of serotransferrin may be involved in iron accumulation in cholangiocarcinoma tissues. ► Carbonylation of serotransferrin, HSP70.1, and A1AT correlated with a poor prognosis.

Section snippets

Subjects

This study was approved by the Ethics Group of the Human Research Committee (HE480316) of Khon Kaen University, Thailand. CCA tissues were obtained with informed consent from patients who underwent surgical resections at Srinagarind Hospital, Khon Kaen University, between 2003 and 2008. Specimens from 36 cholangiocarcinoma patients (18 well differentiated, 4 moderately differentiated, and 1 poorly differentiated tubular adenocarcinoma cases and 13 papillary adenocarcinoma cases) were assessed

Identification of carbonylated proteins by 2D OxyBlot and MALDI–TOF/TOF MS

Fig. 1 shows the 2D-gel electrophoresis with CBB staining of total protein and 2D OxyBlot analysis of carbonylated protein isolated by the DNPH method for each pooled sample (whole (Fig. 1A) and magnified (Fig. 1B) images). 2D OxyBlot analysis detected about 40 carbonylated proteins. Carbonylation was greater in the tumor tissues than in nontumor and normal liver tissues. We performed additional analyses of six proteins of interest, as shown in Table 1, using immunoprecipitation for

Discussion

Our results indicate that carbonylated proteins were more highly accumulated in CCA tissues than in nontumor and normal liver tissues. Induction of an oxidant-generating system and reduction of an antioxidant system are well-recognized causes of oxidative stress. Oxidant-generating enzymes such as inducible nitric oxide synthase and cyclooxygenase 2 were found to be upregulated in animals with O. viverrini-induced CCA, in vitro cell lines, and clinical samples of CCA [22], [23], [24]. Moreover,

Conclusion

The carbonylation of serotransferrin, HSP70.1, and A1AT induced by inflammation may play critical roles in the carcinogenesis of CCA. Oxidative stress induced by inflammation causes the carbonylation of serotransferrin, leading to the accumulation and release of iron. The increased LIP may mediate Fenton reactions and generate ROS, which may contribute to all steps of CCA carcinogenesis. In addition, the carbonylation of HSP70.1 and A1AT may induce protein dysfunction and contribute to the

Acknowledgments

We thank the cadaver donors and their families as well as all of the CCA patients who donated specimens for this study. This work was partly supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants 15406027, 19659150, and 22390121), the Khon Kaen University Research Fund (Grant 49-03-1-01-3), and the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (to R. Thanan and P. Yongvanit).

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    Present address: Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan.

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