The aim of the present study was to use an enzyme-linked immunosorbent assay (ELISA) to determine the concentrations of Lifeguard (LFG) protein in the serum of 36 patients diagnosed with breast cancer and to compare these values with the concentrations of LFG protein in the serum of 7 healthy volunteers in order to detect a possible association between the expression of LFG in the serum and the degree of malignancy of the disease. Although there is no direct association between the LFG protein concentration in the serum and the degree of malignancy of breast cancer, a statistically significant distribution of the concentration in all investigated samples was observed. This indicated an association between the LFG protein concentration in human serum with a currently unknown factor.
In 1785, Johann Heinrich Jänisch created one of the first studies of a disease that triggers up to 14 million new cases worldwide per year: Cancer (
In previous years, the protein Lifeguard (LFG) has been associated with breast cancer (
Furthermore, it was demonstrated that LFG is regulated by the lymphoid enhancer-binding factor 1 (LEF-1) transcription factor, whereas LEF-1 is activated via the phosphoinositide 3-kinase (PI3K)/Akt pathway and interacts with membrane-bound phosphatidylinositol lipids that are also involved in the phosphorylation and activation of Akt kinase (
From April 2015 until May 2016, samples from breast cancer patients and healthy volunteers were collected in association with Professor Lück (Gynecologic Oncology Practice, Hanover, Germany), and the concentration of LFG protein in serum was assessed. Approval for the present study was obtained from the Hannover Medical School's institutional review board (Hanover, Germany). Informed consent was provided according to the Declaration of Helsinki. The study participants included 36 patients with breast cancer and 7 healthy volunteers. Informed consent was obtained from all subjects.
The concentration of LFG protein in the serum of the patients and volunteers was assessed with a FAIM2 ELISA kit (MyBioSource, Inc., San Diego, CA, USA), which was used in accordance with the manufacturer's protocol. A double determination was performed on 36 patients. In addition, 14 wells of a 96-well plate were filled with the serum of healthy volunteers. All sera were stored at −20°C prior to use. A positive control of 150 ng/µl pure LFG protein was added to 2 wells. In order to accurately identify the concentration, a standard was created according to the manufacturer's protocol for the ELISA kit, which may indicate a concentration between 39,063 and 2,500 pg/ml. Following addition of all the samples and a subsequent 2 h incubation at 37°C, a biotin antibody was added and again incubated for 1 h at 37°C. Subsequent to several washing steps, horseradish peroxidase-avidin (eBioscience, Inc., San Diego, CA, USA) was added to each well and incubated for 1 h at 37°C. Subsequent to washing, 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was added to each well and incubated in the dark for 30 min at 37°C. Finally, this was followed by the measurement of the fluorescence signals of the plate at a wavelength of 450 nm with a GENios plate analyzer (Tecan, Männedorf, Switzerland).
The data obtained were analyzed with Origin software version 9 (OriginLab Corporation, Northampton, MA, USA). All experiments were performed in triplicate and repeated at two independent time points. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
Measurement with the ELISA kit was performed to assess whether an association exists between the concentration of LFG protein in the serum of patients and characteristic properties of breast cancer. Therefore, the concentration of LFG protein in the serum of 36 patients diagnosed with breast cancer and the concentrations in the serum of 7 healthy volunteers were assessed. The measurement of LFG serum concentration was performed by detecting the optical density (OD) of the serum samples. The OD450 values are shown in
In order to convert the OD450 values in the corresponding concentrations, the preparation of a calibration curve based on the standard was necessary. This is shown in
where c was the concentration in ng/ml and OD450 was the OD measured at a wavelength of 450 nm.
Using the calibration curve, the serum LFG concentrations of patients and healthy subjects was determined. The OD values for the 36 patients are reported in
The resulting calculated statistical values are listed in
The interval width Δx was derived by dividing the breadth by the number of intervals, as follows:
Thus, the value range was divided into 6 intervals on an identical length of 0.05 ng/ml. These are shown in
A similar result is shown in the histogram of the healthy volunteers. The interval with the highest frequency was, however, interval 2. None of the measured values lay in intervals 1 or 4. The remaining intervals 3, 5 and 6 reflect an equal distribution of 14.3%.
Additional evaluation of the measured values was performed as a function of multiple attributes of the patients and breast cancer. These characteristics included: Age of the patients; type of histological finding; presence of various growth factors, consisting of progesterone and estrogen receptors, and human epidermal growth factor receptor 2; Ki67 value; grade of the tumor; and size of the tumor and presence of lymph node involvement, as well as metastases.
In
Due to the extremely high standard deviation, as well as the similar average values in almost all intervals, a uniform distribution of LFG protein concentration in all investigated ages may be assumed.
In
In
In
In
In the box plots of
The LFG protein concentration in the serum of patients is presented in
Despite the results of the histograms,
The present results demonstrate that a discrete concentration of LFG protein is present in the human serum. Taking into account that LFG is a transmembrane protein and, accordingly, assumes a large extent of cellular functions, this is a significant identification.
Using the ELISA kit, a direct association between the LFG protein concentrations in the serum of patients and various characteristics of breast cancer was excluded. Thus, the initial hypothesis to use the LFG protein concentration in the serum as a tumor marker for the diagnosis of breast cancer cannot be realized. However, the histograms demonstrate distinct maxima, which indicate an association with a currently unknown factor. Thus, further investigation is required. For example, additional examination of the association between other medical conditions, such as rheumatoid arthritis or diabetes, as well as everyday habits such as diet and smoking as aspects associated with LFG protein concentration in the serum. Therefore, additional research on the origin of the LFG protein concentration in the serum may provide important insights into the human biological function and operation of the LFG protein.
The present study was funded by the Niedersächsische Krebsgesellschaft (Hanover, Germany; grant number B/Sc). The authors are grateful to Ms. Andrea Lazaridis (Hanover Medical School, Hanover, Germany) for her excellent technical assistance.
Calibration curve for converting the OD450 values at the respective concentration. OD, optical density.
Box plot of the concentrations of Lifeguard protein in the serum of patients.
Box plot of the concentrations of Lifeguard protein in the serum of healthy volunteers.
Histogram of the concentration of Lifeguard protein in the serum of patients.
Histogram of the concentration of Lifeguard protein in the serum of healthy volunteers.
Measured concentration of Lifeguard protein depending on the age of the patients.
Measured concentration of Lifeguard protein depending on the histological findings of breast cancer.
Measured concentration of Lifeguard protein, depending on the occurrence of estrogen-, progesterone- and HER2-receptors. HER2, human epidermal growth factor receptor 2
Measured concentration of Lifeguard protein as a function of Ki67 value of the tumor (in %).
Measured concentration of Lifeguard protein depending on the grade of the tumor.
Measured concentration of Lifeguard protein depending on the appearing of metastasis, lymph node involvement and tumor size.
OD450 values of the standards, healthy volunteers and a positive control
Standard (OD450) | Standard (OD450) | Healthy (OD450) | Volunteers | Positive (OD450) | Control |
---|---|---|---|---|---|
0.000 | 0.1258 | 0.1763 | 0.2232 | 0.1442 | 0.134 |
39.063 | 0.2423 | 0.4207 | 0.2790 | ||
78.125 | 0.3427 | 0.2658 | 0.2600 | ||
156.250 | 0.4328 | 0.4259 | 0.4245 | ||
312.500 | 0.5058 | 0.1657 | 0.1740 | ||
625.000 | 1.1678 | 0.1676 | 0.1818 | ||
1,250.000 | 2.0601 | 0.1912 | 0.2084 | ||
2,500.000 | 2.5811 |
In order to convert the OD450 values into the corresponding concentrations, the preparation of a calibration curve based on the standard protein concentration was necessary. The conversion factor of OD450 to concentration (ng/ml) was performed using the following equation: Concentration of sample (ng/ml) = OD450 × (0.84 ± 0.073)/50. OD, optical density.
OD450 values of 36 patients
Patient samples (OD450) | ||||
---|---|---|---|---|
0.2473 | 0.2823 | 0.1807 | 0.1608 | 0.2855 |
0.2168 | 0.3487 | 0.1415 | 0.164 | 0.2764 |
0.3808 | 0.4610 | 0.1675 | 0.2546 | 0.1293 |
0.4523 | 0.4558 | 0.2197 | 0.2476 | 0.1436 |
0.2194 | 0.3015 | 0.3447 | 0.1992 | 0.2694 |
0.1945 | 0.3807 | 0.3127 | 0.2507 | 0.1210 |
0.2007 | 0.1553 | 0.2719 | 0.1464 | 0.1327 |
0.2077 | 0.1698 | 0.1314 | 0.1250 | 0.1307 |
0.1389 | 0.1519 | 0.1998 | 0.1397 | |
0.1571 | 0.1721 | 0.1638 | 0.1077 | |
0.2967 | 0.2155 | 0.3027 | 0.1496 | |
0.3198 | 0.3963 | 0.2724 | 0.1127 | |
0.2324 | 0.1304 | 0.2471 | 0.1763 | |
0.2406 | 0.1562 | 0.2301 | 0.2331 | |
0.2267 | 0.1329 | 0.1573 | 0.1142 | |
0.2206 | 0.1584 | 0.1624 | 0.1074 |
Lifeguard serum concentration determination was performed by detecting the OD of the serum samples. OD, optical density.
Concentrations of Lifeguard protein in 36 patients with the standard deviation.
Patient samples, ng/ml | ||||
---|---|---|---|---|
0.196±0.017 | 0.266±0.023 | 0.136±0.012 | 0.137±0.012 | 0.237±0.021 |
0.351±0.030 | 0.386±0.033 | 0.163±0.014 | 0.212±0.018 | 0.115±0.010 |
0.174±0.015 | 0.287±0.025 | 0.277±0.024 | 0.190±0.016 | 0.164±0.014 |
0.172±0.015 | 0.137±0.012 | 0.170±0.015 | 0.114±0.010 | 0.111±0.010 |
0.125±0.011 | 0.137±0.012 | 0.153±0.013 | 0.104±0.009 | |
0.260±0.023 | 0.258±0.022 | 0.242±0.210 | 0.110±0.009 | |
0.199±0.017 | 0.121±0.011 | 0.201±0.017 | 0.172±0.015 | |
0.188±0.016 | 0.123±0.011 | 0.135±0.012 | 0.093±0.008 |
Concentrations of Lifeguard protein in 7 healthy volunteers, as well as the positive control with the respective standard deviation.
Healthy volunteers, ng/ml | Positive control, ng/ml |
---|---|
0.168±0.015 | 0.117±0.009 |
0.295±0.026 | |
0.221±0.019 | |
0.358±0.031 | |
0.143+0.012 | |
0.147±0.013 | |
0.168±0.015 |
Statistical parameters of two measurements of the mean concentration value of Lifeguard protein (all data are provided in ng/ml).
Group | Mean | Standard deviation | Media | Minimum | Maximum | Number |
---|---|---|---|---|---|---|
Patients | 0.184 | 0.070 | 0.171 | 0.093 | 0.386 | 36 |
Healthy individuals | 0.214 | 0.083 | 0.168 | 0.143 | 0.358 | 7 |
Interval breadths in the histograms.
Measurement | Interval 1 | Interval 2 | Interval 3 | Interval 4 | Interval 5 | Interval 6 |
---|---|---|---|---|---|---|
Breadth, ng/ml | 0.09–0.14 | 0.14–0.19 | 0.19–0.24 | 0.24–0.29 | 0.29–0.34 | 0.34–0.39 |