Original article
Experimental validation of the complement protein C3a down expression in the plasma of patients with squamous cell carcinoma of the penis

https://doi.org/10.1016/j.urolonc.2017.05.002Get rights and content

Highlights

  • To validate down expression of complement protein C3a in the plasma of patients with penile cancer with a commercially available ELISA test.

  • Complement system fragment C3a is down expressed in patients with SCCP.

  • These results suggest that the innate immune response might be suppressed in patients with these malignancies.

  • Perhaps in the future, the expression of C3a can be used as a useful marker for penile tumors and other types of cancer.

Abstract

Objectives

We have previously shown the importance of the complement system in differentiating between patients with squamous cell carcinoma of the penis (SCCP) and controls. These patients had low expression of C3a and C4 fragments. Therefore, in this study, we investigated the complement protein C3a as a potential circulating biomarker in these patients by a commercially available enzyme-linked immunosorbent assay (ELISA) test.

Patients and methods

Plasma samples from 39 patients with SCCP, 15 patients with prostate cancer, and 50 healthy male subjects were evaluated using the ELISA—Bioscience OptEIA Kit human anti-C3a (BD). The nonparametric Mann-Whitney test was used for comparison of means among the groups.

Results

The complement protein C3a was found down expressed in patients with SCCP (P<0.05) in comparison to either subjects with good health or subjects with prostate cancer.

Conclusion

Experimental validation of the down expression of C3a was well succeeded using a commercial ELISA kit. Complement system fragment C3a is down expressed in patients with SCCP. Besides, C3a is also low expressed in the plasma of patients with initial prostate cancer when compared to healthy subjects. These results suggest that the innate immune response might be suppressed in patients with these malignancies.

Introduction

Squamous cell carcinoma of the penis (SCCP) represents 95% of the penile cancers. SSCP is considered a rare disease in developed countries. On the contrary, in underdeveloped and emerging countries, SSCP is a neglected type of cancer, reaching up to 10% of male neoplasms in some areas [1], [2]. The incidence of carcinoma of the penis varies according to circumcision practice, hygienic standard, phimosis, number of sexual partners, human papillomavirus infections, exposure to tobacco products, and other factors [2], [3]. Thus, inflammatory processes can be directly associated with this type of cancer [4].

Metastases from penile carcinoma usually spread through penile lymphatic vessels to regional nodes. Recently, our group identified, applying the proteomic approach based on ClinProt platform, 2 differentially expressed peptides comparing the C8 MB eluted fraction of patients׳ vs. control subjects׳ plasma, that corresponded to the fragments of the C3a (m/z 1896.17) and C4 (m/z 2021.26) complement protein. Our results demonstrated that when the disease progresses, the fragments of C3a and C4 become more underexpressed [5]. These fragments seem to be mainly down-regulated in patients with metastatic involvement, and the innate immune response of patients could be suppressed [6]. Given these results, we decided to experimentally validate the expression of C3a in plasma of patients with SCCP comparing with results of plasma levels from patients with prostate cancer (PC) and from control cancer-free subjects, using an enzyme-linked immunosorbent assay (ELISA), which is a more affordable and low-cost test compared to the proteomic approach (mass spectrometry) used in discovery phase. Validation by the ELISA method is important because none of the proteomics methods are 100% safe and deviations may occur. In addition, the method that was used in the previous article is an expensive experimental method that does not apply to the laboratory examination routine. If the intention is to use C3a as routine marker, it must be validated using a routine method such as ELISA.

Section snippets

Study population

The study involved a total of 104 human plasma specimens. First, we tested samples from 39 patients with penile cancer comparing them with 50 samples from healthy cancer-free male volunteers. In addition, 15 samples from patients with PC were also tested comparing them with samples from patients with penile cancer and samples from healthy controls. The mean age was 63 for patients with penile cancer and 67 for patients with PC. The mean age of healthy controls was 60 years. Patients and

Results

Our results showed that the C3a plasma levels concentration (ng/ml) of the control group showed statistically significant difference when compared with plasma from patients with SCCP with Mann-Whitney test. When these patients were stratified into 3 stage groups, the concentrations also showed significant difference (Table 3).

Control group showed a higher median protein concentration (11.93 ng/ml) than that of the 3 stage groups (2.63, 3.04, and 2.69, respectively). Although the comparison

Discussion

The link between cancer and inflammation was first proposed by Rudolf Virchow in the 19th century [8] Virchow observed that long-term inflammation established an environment that promoted the initiation and growth of malignancy [9]. Complement is a central part of the innate immunity that serves as a first line of defense against foreign and altered host cells [10]. The complement system is composed of plasma proteins produced mainly by the liver or membrane proteins expressed on cell surface.

Conclusion

Experimental validation of the down expression of C3a was well succeeded using a commercial ELISA kit. C3a is truly down-regulated in the plasma of patients with SCCP, being a strong circulating biomarker candidate. Besides, C3a is also low expressed in the plasma of patients with PC when compared to healthy subjects. These results suggest that the innate immune response might be suppressed in patients with these malignancies. Down expression of C3a may be important in other types of cancer,

References (21)

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    The authors also reported a significant correlation between higher ARID1A expression and worsening histologic grade, but there was no association with the risk of lymphatic spread and survival-related outcomes. The complement protein C3a was also investigated as a potential circulating biomarker in 39 patients with SCC of the penis by a commercially available enzyme-linked immunosorbent assay test using patients with prostate cancer or healthy male subjects as control [39]. The complement protein C3a was found down expressed in patients with penile SCC (P<0.05) in comparison to either subjects with good health or subjects with prostate cancer.

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    In contrast, some studies showed decrease in serum C3 levels. For example, in patients with squamous cell carcinomas [81] and colorectal cancer [82] downregulated C3 (or C3a) expression was observed. Importantly, in addition to host cells, tumor cells can also produce complement proteins.

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This work was financially supported by Programa de Oncobiologia and FAPERJ (APQ1 E-26/111.336/2013), Rio de Janeiro, Brazil.

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