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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 1  |  Page : 94-98

Expression and clinical significance of centrosomal protein 55 in T-cell lymphoma


Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Xin Wang
No. 324, Jingwu Road, Jinan, 250021, Shandong
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_758_17

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 > Abstract 

Context: T-cell lymphomas (TCLs) have been heterogeneous lymphoid malignancies with aggressive clinical phenotype and poor prognosis. Centrosomal protein 55 (CEP55) played a critical role in cytokinesis and served as a centrosome- and midbody-associated protein. Previous studies have reported the overexpression and clinical significance of CEP55 in various human malignancies, but the exact biological roles of CEP55 in TCLs remained unclear.
Aims: In this study, we aimed to evaluate the CEP55 expression in patients with TCL and reactive hyperplasia of lymph nodes. The correlation between CEP55 levels and clinical characteristics was also explored for TCL patients. For further investigation, the cell viability of TCL cell lines after CEP55 inhibition was also assessed.
Subjects and Methods: Immunohistochemistry was applied to assess the elevated level of CEP55 in TCLs. After siRNA treatment, cell viability and apoptotic rate of TCL cell lines were observed with CCK-8 assay and flow cytometry, respectively.
Statistical Analysis: The Pearson's Chi-square test or Fisher's exact test was applied to analyze the correlations between CEP55 overexpression and clinical characteristics. All statistical tests were two-sided, and P < 0.05 was considered to be statistically significant.
Results: CEP55 was upregulated in TCL patients and significantly correlated with Ki-67 label index. Consistently, cell viability was decreased, and apoptosis was increased after the suppression of CEP55 in TCL cell lines.
Conclusions: These results suggested that target CEP55 would be a novel therapeutic strategy for the TCL.

Keywords: Cell viability, centrosomal protein 55, Ki-67, T-cell lymphoma


How to cite this article:
Xu Y, Zhou X, Li Y, Zhang Y, Wang X. Expression and clinical significance of centrosomal protein 55 in T-cell lymphoma. J Can Res Ther 2018;14:94-8

How to cite this URL:
Xu Y, Zhou X, Li Y, Zhang Y, Wang X. Expression and clinical significance of centrosomal protein 55 in T-cell lymphoma. J Can Res Ther [serial online] 2018 [cited 2019 Nov 12];14:94-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/1/94/226760


 > Introduction Top


T-cell lymphomas (TCLs) have been heterogeneous diseases of lymphoid malignancies. TCLs appeared to be more aggressive with a worse prognosis compared with that of B-cell lymphomas.[1],[2] However, the etiology and uniformed therapeutic regimen remained to be explored. Increasing number of studies showed that deregulations of signaling pathways, including PI3K/AKT pathway, were critical events in TCLs pathogenesis.[3],[4] Therefore, exploring and unveiling specific molecules contributing to signaling pathways and tumorigenesis has become an attractive topic for its significance in the treatment of TCLs.

Centrosomal protein 55 (CEP55) has been identified and characterized a decade ago. CEP55 was a centrosome- and midbody-associated protein and played a crucial role in cytokinesis, during which two daughter cells were separated physically then finishing the cell division process.[5] CEP55 overexpression has been identified in mRNA microarray expression profiles of many human cancers, resulting in the accumulation of unstable multinucleated cells, which tended to tumor genesis.[6] Previous studies have shown that overexpression of CEP55 was associated with increased cell migration and invasion, whereas apoptosis was significantly increased with the inhibition of CEP55.[7]

The analysis on various tumors revealed that several pathways were dysregulated due to abundant CEP55.[8],[9] One of the major prosurvival pathways involving CEP55 was the PI3K/AKT pathway. PI3K phosphorylated phosphatidylinositol 4,5-bisphosphate (PIP2), converted PIP2 to phosphatidylinositol 3, 4, 5 trisphosphate (PIP3), and then stimulated the activation of AKT.[10],[11] AKT played diverse roles in cell cycle, angiogenesis, and cell survival. Overactivation of AKT was mainly achieved through the mutation of PIK3CA in multiple cancers.[12] CEP55 was reported to bind PIK3CA, and AKT activation would be enhanced by their interaction.[4]

This study was designed to investigate CEP55 expression in TCL, as well as exploring the correlation between CEP55 levels in tumor tissues and clinical characteristics. Furthermore, we demonstrated that CEP55 inhibition was significantly correlated with decreased cell viability and increased cell apoptosis. Our results provided a novel therapeutic target and prognosis factor in TCL.


 > Subjects and Methods Top


Patients and samples

The paraffin-embedded tissue samples from 46 TCLs patients without pretreatment and 21 reactive hyperplasia of lymph nodes (RHLs) were enrolled in this study. They were collected from Shandong Provincial Hospital affiliated to Shandong University. All the patients were diagnosed according to the WHO criteria from 2011 to 2015 including 11 cases of peripheral TCL nonspecific (PTCL-NOS), 9 cases of angioimmunoblastic TCL (AITL), 6 cases of T-cell lymphoblastic lymphoma (TLBL), 3 cases of enteropathy-associated TCL (EATL), and 9 cases of natural killer (NK)/T-cell lymphoma (NK/TCL). This study was approved by the Medical Ethical Committee of Shandong Provincial Hospital affiliated to Shandong University. All human samples were obtained after informed consents were obtained, according to the Declaration of Helsinki.

Cell culture

In this study, three T-cell lines were included: Jurkat (T-cell acute lymphoblastic leukemia cell line), Karpass299 (ALK + ALCL cell line), and Myla3676 (Cutaneous T-cell lymphocyte, lymphoblast, Sezary syndrome). Jurkat and Karpass299 were obtained from the Typical Culture Preservation Commission Cell Bank (Chinese Academy of Sciences, China) and Shanghai Bioleaf Biotech Company Limited, respectively; Myla3676 was retained by our laboratory.[13] Three cell lines were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA), 1% penicillin/streptomycin mixture, and 2 mM L-glutamine at 37°C in humidified atmosphere containing 5% CO2.

Immunohistochemistry

Paraffin-embedded tissue sections were deparaffinized and hydrated. High-pressure antigen retrieval was then performed in 0.01 M sodium citrate (PH 6.0). Followed by incubating with primary rabbit antibodies for CEP55 (Abcam, USA) overnight at 4°C in a humidified chamber, blockage of endogenous peroxidase and nonspecific staining was performed with 3% H2O2 and normal serum, respectively. The sections were then incubated with the second antibody from SP reagent kit (Zhongshan Goldenbridge Biotechnology Company, China). The sections were stained with hematoxylin and mounted after developing with diaminobenzidine kit (DAB, Zhongshan Goldenbridge Biotechnology Company, China). Each section was assessed in a series of randomly selected 5 high-power fields at ×400 magnification by two independent observers who were blinded to all clinical data. The sections were scored according to the proportion of positively stained tumor cells, where more than 30% of the positively stained cells were categorized as positive cases.

Establishment of siRNA-treated cells

Cells were seeded into 24-well plates (2 × 105 cells/well). Either siRNA targeting CEP55 or negative control siRNA (RIBOBIO, China) was added to each well (100 nM each) with transfection reagent (RIBOBIO, China). After incubating for 24 h, the cells were applied for the indicated experiments.

Western blot

For western blot assays, total protein was extracted from cells after the indicated treatment. Cells were washed for three times in cold phosphate-buffered solution (PBS) and lysed in lysis buffer (Solarbio, China) containing 1% PhosSTOP phosphatase inhibitor (Roche, Switzerland). Bicinchoninic acid assay (Solarbio, China) was applied to measure protein concentration. Primary antibodies against CEP55 were purchased from Abcam. β-actin was applied as the loading control. Primary antibody against β-actin was obtained from Zhongshan Goldenbridge Biotechnology Company. Horseradish peroxidase-conjugated goat-anti-mouse and goat-anti-rabbit antibodies (Zhongshan Goldenbridge Biotechnology Company, SPN-9002 and SPN-9001, respectively) were involved as secondary antibodies.

Cell counting Kit-8 assay

Cells were seeded into 96-well plates (1 × 104 cells/100 μL/well, 37°C, 5% CO2) for 6, 12, 24, and 48 h, respectively. The viability was assessed with the Cell Counting Kit-8 (CCK-8; Dojindo, CK04). Thereafter, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for 2 h (37°C, 5% CO2). The absorbance at 450 nm (reflected cell viability) was read with a SpectraMax M2 multimode microplate reader (Molecular Devices, USA).

Assessment of cell apoptosis

Cell apoptotic rate was evaluated with annexin V-phycoerythrin (PE)/7-amino-actinomycin D (7-AAD) assay (BD Biosciences, USA) through flow cytometry. 1 × 106 cells were harvested after designed treatments and resuspended in 100 μL of 1X binding buffer after washed twice with cold PBS, to which 5 μL of annexin V-PE and 5 μL of 7-AAD were added. The fluorescence of the stained cells was measured immediately with a Navios flow cytometer (BECKMAN COULTER, USA). Samples individually stained for annexin V-PE and 7-AAD were applied to adjust the compensation. All experiments were performed in triplicate and repeated three times.

Statistical analysis

Results were represented as mean ± standard error of mean and each cytological experiment with cell lines was repeated three times. The Pearson's Chi-square test or Fisher's exact test when necessary was applied to analyze correlations between CEP55 overexpression and clinical characteristics. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.


 > Results Top


Elevated expression level of centrosomal protein 55 in T-cell lymphoma tissues

Expression of CEP55 in 46 cases of TCLs was determined by immunohistochemistry, compared with that of in the control of 15 cases of RHL. CEP55 staining was readily observed in most of TCLs samples, but it was hard to be detected in RHL tissues due to weak staining [Figure 1]. Samples displaying 30% or more stained cells were categorized as positive cases. The positive cases of CEP55 staining in PTCL-NOS, AITL, TLBL, EATL and NK/TCL were 8 (72.73%), 5 (55.56%), 12 (85.71%), 2 (66.67%), and 7 (77.78%), respectively. More detailed data were summarized in [Table 1].
Figure 1: Centrosomal protein 55 was overexpressed in T-cell lymphoma tissues analyzed with immunohistochemistry. Representative IHC staining of Centrosomal protein 55 in reactive hyperplasia of lymph nodes, peripheral T-cell lymphoma-not otherwise specified, Angioimmunoblastic T-cell lymphoma, T-cell lymphoblastic lymphoma, Enteropathy-associated T-cell lymphoma, and natural killer/T-cell lymphoma, respectively

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Table 1: Immunohistochemical expressions of centrosomal protein 55 in T-cell lymphoma tissues

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Correlation between centrosomal protein 55 expression and clinical features

The correlation between CEP55 expression and clinical characteristics was shown in [Table 2]. The results of Pearson's Chi-square test or Fisher's exact test when necessary indicated that CEP55 expression was significantly associated with Ki-67 (P = 0.0171). No other clinical parameter was detected to be associated with CEP55 overexpression.
Table 2: Association between centrosomal protein 55 expression level and clinical characteristics in T-cell lymphoma tissues

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Inhibition of centrosomal protein 55 impaired the biological behavior in T-cell lymphoma cell lines

In Jurkat, Karpass299 and Myla3676 cell lines, <80% of cells were successfully transduced with siRNA targeting CEP55. CEP55 expression was downregulated, which was confirmed by Western blotting [Figure 2]a. Cell viability was observed in 6, 12, 24, and 48 h, respectively. Significant decrease of cells viability was observed in siCEP55-interfered cells after incubating for 48 h, compared with that of in cells treated with siNC [Figure 2]b. Consistently, suppression of CEP55 increased cell apoptosis rate in three cell lines [Figure 2]c.
Figure 2: Centrosomal protein 55 was essential for the proliferation of T-cell lymphoma cell lines. (a) Western blot showed centrosomal protein 55 expressions in Jurkat, Karpass299, and Myla3676 cells transfected with centrosomal protein 55 siRNA. β-Actin was served as an internal control. (b) Cell Counting Kit-8 assay showed that depletion of centrosomal protein 55 significantly suppressed the proliferation and viability of T-cell lymphoma cells. (c) Flow cytometry of apoptosis assay indicated increased apoptotic rate after centrosomal protein 55 interference in Jurkat, Karpass299, and Myla3676. *P < 0.05; **P < 0.01. P < 0.05 was considered statistically significant

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 > Discussion Top


TCLs accounted for roughly 10%–15% of lymphoid malignant diseases.[14] The incidence of TCLs varied geographically, with the highest prevalence in some regions of Asia.[15] For some subtypes of TCLs such as mycosis fungoides or CD30+ cutaneous lymphoproliferative disorders, the prognosis may be better prognosis and disease history may be longer. However, TCLs with aggressive phenotype were always correlated with a short survival. It has been widely acknowledged that specific genetic alterations as well as aberrant signaling pathways were pivotal events in the initiation and progression of TCLs.

Upregulation of CEP55 has been identified in a number of human malignant diseases and cancer cell lines.[16],[17] The wide distribution of high CEP55 level among various cancers made it a promising target for cancer target therapy. However, the expression profile and exact roles of CEP55 in tumorigenesis and development of TCL still required clarification. Therefore, in this study, the protein expression profile of CEP55 in TCL was analyzed. To our knowledge, this has been the first research to investigate the possible roles of CEP55 in TCL.

In the present study, we have detected upregulated CEP55 protein expression in 73.91% (34/46) patients with immunohistochemistry. The positive cases of CEP55 in PTCL-NOS, AITL, TLBL, EATL, and NK/TCL was 8 (72.73%), 5 (55.56%), 12 (85.71%), 2 (66.67%), and 7 (77.78%), respectively. Our results were consistent with that of previous studies, which reported high CEP55 levels in various tumor tissues compared to that of nontumorous tissues.[9],[16],[18] Furthermore, the expression of CEP55 was significantly correlated with Ki-67 levels. Physiologically, CEP55 was only overexpressed in thymus, testis, small intestine, and placenta. It indicated that CEP55 was associated with high proliferation potency of human cells and high levels of CEP55 might facilitate the progression of tumors.

Our study further concluded that CEP55 was not only upregulated in TCLs but also involved in the proliferation of TCL cell lines. Herein, inhibition of CEP55 through siRNA would result in significantly augmented cell viability inhibition and apoptosis in vitro, compared to that of in negative control cells.

Although inspiring, there were still some limitations in the present study:First, exact molecular mechanisms on how CEP55 promoted cell proliferation remained unclear. Second, even though CEP55 was overexpressed in TCLs, further validation would be necessary for the clinical application of CEP55.

Taken together, our results showed that CEP55 was upregulated and exerted an oncogenic role in TCL. Our study also suggested that target CEP55 would be a novel strategy in the treatment of TCL.


 > Conclusion Top


This study demonstrated that CEP55 overexpressed in T-cell lymphoma, and CEP55 expression levels in tumor tissues were strongly correlated and clinical characteristics. Furthermore, we investigated that CEP55 inhibition induced significantly decreased cell viability and increased cell apoptosis. On this basis, our results provided a novel therapeutic target and prognosis factor in T-cell lymphoma.

Financial support and sponsorship

This study was supported by the National Natural Science Foundation (No. 81473486 and No. 81270598), the Natural Science Foundations of Shandong Province (No. ZR2012HZ003), the Technology Development Projects of Shandong Province (No. 2014GSF118021), the Program of Shandong Medical Leading Talent, and the Taishan Scholar Foundation of Shandong Province.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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    Figures

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    Tables

  [Table 1], [Table 2]



 

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