Elsevier

Gene

Volume 591, Issue 1, 10 October 2016, Pages 236-244
Gene

Research paper
Downregulation of Peptidylprolyl isomerase A promotes cell death and enhances doxorubicin-induced apoptosis in hepatocellular carcinoma

https://doi.org/10.1016/j.gene.2016.07.020Get rights and content

Highlights

  • We investigated the expression of PPIA in HCC tissues and HCC patients serum.

  • The expression of PPIA was significantly increased in HCC patients, however in serum was down-regulated.

  • Knockdown of PPIA sensitizes HCC cells to doxorubicin.

  • PPIA could serve as a novel marker and therapeutic molecular target for HCC patients.

Abstract

Peptidylprolyl isomerase A (PPIA) is a peptidyl-prolyl cis-trans isomerase that is known to play a critical role in the development of many human cancers. However, the precise biological function of PPIA in hepatocellular carcinoma (HCC) remains largely unclear. In this study, lentiviral overexpression vectors and small interfering RNA knockdown methods were employed to investigate the biological effects of PPIA in HCC. PPIA levels in HCC tissues and peritumoral tissues were detected by real-time Polymerase Chain Reaction (RT-PCR), Western blotting, and immunohistochemistry. Our results indicate that PPIA levels were significantly higher in the HCC tissues compared to the matched peritumoral tissues. Moreover, PPIA expression was significantly associated with tumor size in these tissues. Interestingly, serum PPIA (sPPIA) levels were significantly higher in healthy controls compared to the HCC patients. Knockdown or overexpression of PPIA was shown to downregulate and upregulate cell growth, respectively. Moreover, PPIA siRNA knockdown appears to promote doxorubicin-induced apoptosis in HCC cells, altering the expression of downstream apoptotic factors. In summary, our results indicate that PPIA may play a pivotal role in HCC by regulating cell growth and could serve as a novel marker and therapeutic molecular target for HCC patients.

Introduction

Hepatocellular carcinoma (HCC), one of the major cause of malignant tumor in China, is the fifth-most common cancer and the third cause of cancer-related mortality worldwide (Siegel et al., 2014). The relationship between chronic infection with hepatitis B virus and hepatocellular carcinoma is well established, and some 340,000 cases of HCC are attributable to hepatitis B infection (Parkin, 2006). The clinical characteristics of HCC include highly aggressive, recurring tumor formation that are frequently associated with metastasis to the lungs. To treat HCC patients, surgical resection, chemoembolization, radiation therapy, and liver transplantation have all been employed. However, even with these techniques, the incidence rate is still highly correlated to the death rate, with the five-year survival rate only being modestly improved to approximately 26% in the United States (Maluccio and Covey, 2012, Simard et al., 2012). Although progress has been made in recent decades to better understand and treat HCC, the underlying molecular mechanisms of this aggressive cancer remain elusive (El-Serag and Rudolph, 2007). Therefore, it is critical to explore the cellular signaling cascades operating during HCC pathogenesis in order to provide insights into other diagnosis and treatment options for HCC patients.

Peptidylprolyl isomerase A (PPIA), also known as CYPA, a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family, catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerates protein folding. It was originally purified from bovine thymocytes and characterized as the primary cytoplasmic binding protein of the immunosuppressant cyclosporin A (CsA) (Handschumacher et al., 1984). Previous studies have also reported that PPIA is involved in several diseases, including viral infection, cardiovascular disease, and various inflammatory diseases (Franke et al., 1994, Jin et al., 2000, Gwinn et al., 2006). Moreover, this enzyme has also been shown to be a key molecule in multiple biological functions, including molecular chaperoning, protein folding, protein trafficking, immune modulation, and cell signaling (Kern et al., 1995, Galigniana et al., 2004, Syed et al., 2003, Colgan et al., 2004). Phosphorylation of apoptosis signaling-regulating kinase 1 (ASK1) is also regulated by PPIA, which reduces the expression and function of ASK1 as well as its downstream kinases in the JNK and p38 signaling pathways (Kim et al., 2015). Recently, several studies have shown that PPIA is overexpressed in some human cancers, including non-small cell lung cancer, pancreatic adenocarcinoma, tongue squamous cell carcinoma, and head and neck squamous cell carcinoma (Campa et al., 2003, Howard et al., 2005, Qian et al., 2012, Li et al., 2005, Li et al., 2006, Huang et al., 2012, Takahashi et al., 2012). Furthermore, PPIA also appears to participate in cancer proliferation, cell cycle progression, regulation of apoptosis, and cell migration/invasion, all of which play various pathophysiological functions during tumor progression (Li et al., 2008, Semba and Huebner, 2006, Choi et al., 2007, Calhoun et al., 2009). However, although there is a growing body of evidence that PPIA may play a pivotal role during tumor development, our understanding of the precise roles of PPIA in tumor cell proliferation and apoptosis is limited, particularly in HCC.

In the present study, we sought to determine the clinical significance of PPIA in HCC carcinogenesis and progression. To do so, we first established the expression level of PPIA in HCC tissue, followed by functional analysis in multiple liver cancer cell lines. With these data, we further clarified the molecular relationship between PPIA and HCC progression, during which downregulation of PPIA appears to promote cell death and enhance doxorubicin-induced apoptosis.

Section snippets

Patients

Ninety HCC tissues and peritumoral tissues were randomly selected from patients undergoing hepatectomy between 2012 and 2014. Blood samples were collected between May 2014 and November 2014 from Fifty-four patients with histologically documented HCC who were candidated for surgical treatment. The control group consisted of one hundred and five healthy blood donors. All specimens were from our hospital (First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, China). Sample

Pattern of PPIA expression in HCC tissues and HCC patients serum

To explore the role of PPIA during HCC pathogenesis, we observed its expression in HCC tissues using Western blotting and real-time PCR. It appears that, at the mRNA level, PPIA was overexpressed in the 51 HCC tissue samples studied compared to their matched peritumoral tissues (Fig. 1A). Moreover, our Western blot analysis indicates that this increase in PPIA mRNA also corresponds to higher PPIA protein expression in the HCC tissues compared to their matched peritumoral tissues (Fig. 1B). For

Discussion

PPIA also was known as CYPA, which is an abundantly expressed protein that possesses peptidyl prolyl cis-trans isomerase activity. Recent research has provided compelling evidence identifying important roles for PPIA in many biological processes. For example, PPIA overexpression has been demonstrated in multiple types of cancer and appears to be related to the clinicopathological symptoms of the patients. Notably, upregulation of PPIA was first reported in HCC in 1998 (Corton et al., 1998).

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

This study was supported by grants from the National High Technology Research and Development Program 863 of China (No. 2012AA021002), Science Technology Department of Zhejiang Province (No. 2015C03034), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421062), National S&T Major Project (No. 2016ZX10002020), the National Health and Family Planning Commission of China (No. 2016138643) and Special Fund for Health Research in the Public

References (58)

  • M. Li et al.

    Effect of cyclophilin A on gene expression in human pancreatic cancer cells

    Am. J. Surg.

    (2005)
  • Z. Li et al.

    Proteomics identification of cyclophilin a as a potential prognostic factor and therapeutic target in endometrial carcinoma

    Mol. Cell. Proteomics

    (2008)
  • Y. Li et al.

    Expression and prognostic relevance of cyclophilin A and matrix metalloproteinase 9 in esophageal squamous cell carcinoma

    Diagn. Pathol.

    (2013)
  • Z. Qian et al.

    Downregulation of cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

    BMC Cancer

    (2012)
  • F.M. Syed et al.

    Antigen entrapped in the escheriosomes leads to the generation of CD4(+) helper and CD8(+) cytotoxic T cell response

    Vaccine

    (2003)
  • J. Yang et al.

    Identification of candidate biomarkers for the early detection of nasopharyngeal carcinoma by quantitative proteomic analysis

    J. Proteome

    (2014)
  • M.J. Bouchard et al.

    The enigmatic X gene of hepatitis B virus

    J. Virol.

    (2004)
  • A. Burlacu

    Regulation of apoptosis by Bcl-2 family proteins

    J. Cell. Mol. Med.

    (2003)
  • C.C. Calhoun et al.

    Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype

    Mol. Cell. Biochem.

    (2009)
  • M.J. Campa et al.

    Protein expression profiling identifies macrophage migration inhibitory factor and cyclophilin a as potential molecular targets in non-small cell lung cancer

    Cancer Res.

    (2003)
  • K.J. Choi et al.

    Overexpressed cyclophilin A in cancer cells renders resistance to hypoxia- and cisplatin-induced cell death

    Cancer Res.

    (2007)
  • S. Cory et al.

    The Bcl2 family: regulators of the cellular life-or-death switch

    Nat. Rev. Cancer

    (2002)
  • E.Z. Eisenmesser et al.

    Enzyme dynamics during catalysis

    Science

    (2002)
  • J. Fanghanel et al.

    Insights into the catalytic mechanism of peptidyl prolyl cis/trans isomerases

    Front. Biosci.

    (2004)
  • W. Feng et al.

    Cyclophilin A enhances cell proliferation and xenografted tumor growth of early gastric cancer

    Dig. Dis. Sci.

    (2015)
  • E.K. Franke et al.

    Specific incorporation of cyclophilin A into HIV-1 virions

    Nature

    (1994)
  • S.F. Gothel et al.

    Cyclophilin and trigger factor from Bacillus subtilis catalyze in vitro protein folding and are necessary for viability under starvation conditions

    Biochemistry

    (1998)
  • W.M. Gwinn et al.

    Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention

    J. Immunol.

    (2006)
  • R.E. Handschumacher et al.

    Cyclophilin: a specific cytosolic binding protein for cyclosporin A

    Science

    (1984)
  • Cited by (24)

    • Noncoding RNA-mediated macrophage and cancer cell crosstalk in hepatocellular carcinoma

      2022, Molecular Therapy Oncolytics
      Citation Excerpt :

      Further study found that PPIAP22 may act as a ceRNA by sponging miR-197-3p to relieve repression of its parental gene peptidylprolyl isomerase A (PPIA).184 Similar to PPIAP22, upregulation of PPIA is also associated with tumor size and poor patient survival and has been shown to promote tumor growth.184,230 The expression levels of PPIAP22 or PPIA were correlated with the infiltration levels of tumor-infiltrating immune cells (TIICs), such as T cells, dendritic cells, and macrophages, and with the expression of several chemokines, including C-C motif chemokine ligand 15 (CCL15) and C-X-C motif chemokine ligand 12 (CXCL12).184

    • Tandem mass tag labeling to characterize muscle-specific proteome changes in beef during early postmortem period

      2020, Journal of Proteomics
      Citation Excerpt :

      Other research has also indicated that HSPD1 has a positive correlation with a* value in beef semitendinosus (ST) muscle during the extended storage [130]. Peptidyl-prolyl cis-trans isomerase A (PPIA) has been also shown to have a significant protective function in cell survival [131–133]. Specifically, it promotes cell survival by alleviating the generation and damage of ROS [134–137], activating signal transducer and activating transcription 3 (STAT3; an antiapoptotic transcription factor) [138], promoting autophagy [139], modifying p53 function [140], and increasing expression of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) [141,142].

    View all citing articles on Scopus
    View full text