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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 590-596

Expression of complement C5a receptor and the viability of 4T1 tumor cells following agonist–antagonist treatment


1 Department of Veterinary Pre-Clinical Sciences, Universiti Putra Malaysia, Selangor, Malaysia
2 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine; Institute of BioScience, Universiti Putra Malaysia, Selangor, Malaysia

Date of Web Publication25-Jul-2016

Correspondence Address:
Mohd Hezmee Mohd Noor
Department of Veterinary Pre.Clinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400-Serdang, Selangor
Malaysia
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Source of Support: National Biotechnology Division, Ministry of Science, Technology and Innovation (MOSTI), No: 02.01.04.SF1206,, Conflict of Interest: None


DOI: 10.4103/0973-1482.146066

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 > Abstract 


Background: Complement system is theoretically believed to halt the progression of tumor by the activity of C5a/CD88. Protein C5a is a potent pro.inflammatory mediator that activates the complement system by binding to its receptor.
Objectives: The purpose of this study is to determine the expression of the anaphylatoxin C5a receptor on 4T1 cell line and to study the viability of the cells after being treated with the C5a peptides.
Materials and Methods: The cells 4T1 had undergone immunofluorescence staining, conventional polymerase chain reaction (PCR) and real-time PCR for the expression of determination part. Whereas Alamar Blue and MTT assays were conducted for the viability study of the cells.
Results: The cells showed positive result in expressing the receptor of the C5a through immunostaining and PCR. The CT value recorded at initial dilution was 22.24. In cell viability assay, the cell was treated with C5a peptides, PMX205 and EP54. The purpose of this treatment was to see whether C5a had a direct effect on the cell itself using both assays. The result showed that PMX205, which is an antagonist, gave more effects towards the cell as compared with the treatment of EP54.
Conclusion: This experiment shows the presence of C5a receptor on 4T1 cell line. We believe that the antagonist peptide is eligible to be used widely in cancer immunotherapy field; but in vivo studies need to be carried out first in the future, as it will determine how these drugs affect the tumor cell growth.

Keywords: C5a, C5aR, 4T1, C5a peptides, Tamoxifen


How to cite this article:
Kosni NN, Ganti N, Noor MH, Razak IS, Ajat MM, Omar AR. Expression of complement C5a receptor and the viability of 4T1 tumor cells following agonist–antagonist treatment. J Can Res Ther 2016;12:590-6

How to cite this URL:
Kosni NN, Ganti N, Noor MH, Razak IS, Ajat MM, Omar AR. Expression of complement C5a receptor and the viability of 4T1 tumor cells following agonist–antagonist treatment. J Can Res Ther [serial online] 2016 [cited 2019 Dec 12];12:590-6. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/590/146066




 > Introduction Top


The complement system is a part of defensive system involved in innate immunity. It also connects the innate immunity with the adaptive immune system as it is involved in multiple types of immune cells. Complement systems are able to guard the host's intravascular space by phagocytosis, opsonize and lyze foreign materials. It also has the capability to promote local acute inflammatory response, which later resulted in instructing and influencing the adaptive immune response.[1] The complement system composes of complex series of more than 30 plasma and membrane-associated proteins, which are widely distributed in circulation and tissues.[2] The activation of complement system occurs via several pathways: The classical, mannan-binding lectin (MBL), alternative and extrinsic pathways.[3]

Protein C5 is the initiator of the effector phase of the complement system and shares with C3, C4 and other proteins of the α,2-macroglobulin super-family. There are two molecular structure chains (α,β) that are linked by disulfide bonds, in which the only difference being C5 not containing an internal thio-ester bond as compared to C3 and C4. The C5 molecule is cleaved into C5a and C5b at the position arg751-leu752 on the α-chain by the convertases of the classical/lectin (C4b2a3b) and alternative pathways (C3bBb3b).[4] The C5a anaphylatoxin receptor is a part of the G protein-coupled receptor family involved in chemoattraction and activation of myeloid cells, as well as in host defense against infection.[5] The C5a, a glycoprotein of 74 amino acids (~11kDa) with a four-helix bundle structure, is a potent bioactive molecule that can act upon a wide variety of cell types expressing its high affinity bonding trans-membrane receptors C5aR/CD88 and C5L2.[6] It is also known as pleiotropic molecule, which acts with other complement component to fasten the destruction of pathogens. However, complement activation may as well contribute to the development of inflammation and immunological diseases. Inflammation is a result from the interaction between host and agents such as pathogens, particulate matter and cancer cells, which bring into physiological responses.[7] Acute inflammation can be represented as heat, redness, pain, swelling and loss of function. All the criteria stated were the example results obtained from the complement activation component C5a, which was a potent inflammatory peptides that contained chemoattractant properties for neutrophils, monocytes and macrophages. An excess activation of complement system also could end up with over inflammatory signals produced [8], which later contribute to the development of inflammation-related diseases and cancer. Some examples of experimental inflammatory diseases involved with over activation of complements are sepsis [9], ischemia-reperfusion injuries [10] and inflammatory bowel disease.[11]

According to Markiewski et al.[12], a connection exists between C5a and tumor progression. Myeloid-derived suppressor (MDS) cells, a type of mediated cells, are being recruited to tumors with the aid of protein C5a. The MDS cells are tumor-induced immune suppression derived cells that can cause harmful to the success of cancer immunotherapies field. One of the new methods in conjunction with immunotherapeutics is to treat tumor progression by modulating the usage of protein C5a as a target drug for treating cancer by suppressing the MDS cell population.[13]

In this study, we investigate the expression of anaphylatoxin receptor C5a in the malignant murine mammary cancer cell, 4T1. To date, there has been no published article regarding the C5aR expression on 4T1 cell lines. The only evidence regarding the expression of C5aR in a murine mammary tumor cells was made by Kim et al.[14], but in another type of murine mammary tumor cell line; EMT6 that is known had expressed this receptor. Both types of cells (4T1 and EMT6) are similar, but differ in terms of the aggressiveness and invasiveness of the respective cells. In addition, we also focused on the effects of receptor modulation on cell growth and viability by both C5a agonist and antagonist peptides. PMX205 is a synthetic peptide (hydrocinnamate-[OPdChaWR]), that was used experimentally to reduce the effects of various disorders related with excess activation of C5a in tissues whereas EP54, the conformational biased decapeptide (YSFKPMPLAR), is capable to induce systemic response due to the complement activation.[15],[16]


 > Materials and Methods Top


Cell culture

The mouse mammary tumor cell lines 4T1 was obtained from American Type Culture Collection (ATCC ®, USA) and maintained throughout in RPMI 1640 medium (Invitrogen™, USA) supplemented with heat-inactivated 10% fetal calf serum (FCS) at 37o C in an atmosphere of 5% CO2 in air. The medium was changed after 2–3 days, and the cells were passaged by the process of trypsinization.

Immunoflourescence staining

About 1.0 × 103 cells were seeded on glass coverslips and allowed to grow for 2–3 days before fixation for 10 minutes in 4% paraformaldehyde in phosphate buffered saline (PBS; pH 7.4). The cells were then incubated with rat anti-mouse C5aR/CD88 (Hycult Biotechnology, Netherlands), irrelevant immunoglobulin (negative control) followed by the appropriate secondary antibodies. Coverslips were mounted with Dako fluorescence mounting medium (Agilent Technologies, USA) and observed under the fluorescence microscope.

Polymerase chain reaction (RT-PCR and qPCR)

Total RNA was extracted and purified using Qiagen RNeasy Kit, according to manufacturer's instruction (Qiagen ®, Valencia, CA), followed by DNase treatment 1 (Invitrogen ™, USA) to eliminate possible genomic contamination. To ensure its purity, RNA was tested using spectrophotometer and compared by the ratio of A260/A280. The RNA was stored at − 80°C until ready to use. Primer sequences for C5aR and GAPDH were adapted from the previous studies; 229kb GAPDH-1 (5′_-TGATGACATCAAGAAGGTGGTGAAG-3′_), GAPDH-2 (5′_TTCTTGGAGGCCATGTAGGCCAT-3′_), 409kb C5aR-1 (5′-TAT AGT CCT GCC CTC GCT CAT-3′) and C5aR-2 (5′-TCA CCA CTT TGA GCG TCT TGG-3′). The conventional PCR was then performed with these cycling conditions, 94°C for 5 minutes, followed by 45 cycles of 30 seconds at 94°C, the annealing temperature of 60°C for 30 seconds and extending temperature of 72°C, each for 30 seconds. The non-template control with nuclease-free water instead of RNA template was also included as negative controls. The RT-PCR products were confirmed by gel electrophoresis of samples in 1.5% agarose gel.

For RT-PCR, one-step of GoTaq qPCR Master Mix (Promega Corporation, USA) was used for quantification of PCR product. To check the specificity of the amplified products, a melting curve analysis was performed, as well as gel electrophoresis. All samples were amplified on the same time and condition. To ensure equal amplification and no-template controls with water were also included as negative control. The sample was then prepared in 1:10 dilution factor to observe the expression of C5a receptor on different concentration. The RT-PCR was then conducted by using Applied Biosystem StepOne Machine.

Cell viability assay

Alamar blue assay

To assess the effects of both C5a antagonism and agonism on the ratio of live: Dead cells in 4T1 culture (in growth medium containing FCS) were seeded at 2.5 × 104 cells/mL/well into each well of a 24-well culture dish. After 24 h, growth medium was replaced with serum-free medium for a further 24 h to synchronize the cells before treatment with EP54 or PMX205 at 0.1M (in serum-free medium) for a further 24 h. The live: Dead cell ratio was assessed by incubating the cells with 10 µL Alamar Blue ® dye (Invitrogen™, USA). In this assay, viable cells convert the blue dye to pink that resulted from the reduction process of resazurin, an active ingredient of the dye to resorufin, which later increased the overall fluorescence and color of the medium surrounding cells. The value of the absorbance was depended on the amount of reagent that was taken up by the live cells. The growth and viability of the cells were assessed at different timeline; 0, 24, 48 and 72 h, respectively, at wavelength 570 nm and 600 nm by using ELISA plate reader Infinite ® M200 (TECAN Group Limited, Switzerland).

Owing to overlap in the absorption spectra of the oxidized and reduced form of Alamar blue, an equation derived by Willard [17] and provided by the manufacturer allows calculation of the percentage of reduction:



wherein 117,216 = molar extinction coefficient of Alamar blue in the oxidized form at 600 nm; 80,586 = molar extinction coefficient of Alamar blue in the oxidized form at 570 nm; 14,652 = molar extinction coefficient of Alamar blue in the reduced form at 600 nm; 155,677 = molar extinction coefficient of Alamar blue in the reduced form at 570 nm; A600 = absorbance of test wells at 600 nm; A570 = absorbance of test wells at 570 nm; A'600 = absorbance of negative control wells at 600 nm; A'57O = absorbance of negative control wells at 570 nm.

MTT assay

Cells were plated at a density 5.0 × 104 cells/ml/well of 96-multiwell plates in complete medium. After 24 h of incubation, the medium was changed with serum starve medium to synchronize the cell cycle and growth as to ensure that the cells reaches its plateau phase. After 24 h of serum-starving, cells were treated with 5 μl/well of 0.lM of agonist, EP54, antagonist, PMX205 drug and the positive control was treated with Tamoxifen drug. 5 μl/well of the 12mM ck solution was added 5 h before reading was taken and about 50 μl/well of the SDS-HCL solution was added and mixed thoroughly using pipette 3 h before reading was taken. The MTT assay was assessed at different time point; 0, 24, 48 and 72 h at wavelength 570 nm respectively by using ELISA plate reader Infinite® M200 (TECAN Group Limited, Switzerland).

Statistical analysis

Results were analyzed as mean ± SEM. Statistical analysis were made by using repeated one-way analysis-of-variance (ANOVA) with Dunnett post hoc test to determine the significance difference of the treatments by using the statistical software package Graphpad Prism version 5.0 (GraphPad Software, San Diego, CA). In all statistical analysis, P value of < 0.05 was considered significant.


 > Results Top


Immunoflourescence staining

The results showed that there was an expression of C5a receptors in the 4T1 cell line at the membrane of the cells [as indicated with arrows in [Figure 1]. The greenish color showed the expression of the C5a receptors by the 4T1 cell line.
Figure 1: The expression of C5a receptors in the 4T1 cell lines. 1:100 dilution of secondary antibody was added to the sample whereas for the control group, no antibody was added to the coverslip during the experiment. Coverslips were mounted with Dako fluorescence mounting medium. All the pictures were taken using the 63 × magnification

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PCR

To further extend the results of the expression of C5a receptor in 4T1 cell line, semi-quantitative and quantitative PCR (qPCR) were conducted. [Figure 2] and [Figure 3] summarized the results of these experiments. Semi-quantitative PCR and qPCR studies demonstrated the presence of C5a receptor in 4T1 cell line which consistent with the immunostaining findings. All amplicons were of expected size. The CT value obtained from qPCR showed an increasing value as dilution factors were increased. The initial CT value recorded was about 22.24. The higher the number of CT value, the lower will be the concentration of C5a receptor expressed.
Figure 2: Result of electrophoresis in 1.5% TBE-agarose gel of conventional RT-PCR product by using two set of primers; C5aR Primer with 409 bp and GAPDH primer with 229 bp as a housekeeping gene. Appropriate DNA marker was included in the left hand lane. No band was detected under the NTC (non template control) lane

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Figure 3: Real-time PCR was conducted to obtain the CT value data of 4T1 cell line with stock concentration of 48.7 ug/ml followed by serial dilution of 1:10. The expression of C5a receptor on 4T1 cell line is inversely proportional to the CT value recorded, the higher the CT value recorded; the lower will be the expression of C5a receptor on 4T1 cell line. Data for each bar is expressed as a mean value of CT value reading for each dilution. * denotes the significant values (P < 0.05), in which Dunnett post-hoc test is used in analyzing the data

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Cell viability assay

Alamar blue assay

Due to the metabolic activity in the Alamar blue assay, the assay can be analyzed by using absorbance at 570 nm [Figure 4] or by calculating the percentage of reduction [Figure 5]. Only results at 24, 48 and 72 h at reduced wavelength were shown here. In reduction wavelength, 570 nm, 0 h plate showed significant results in all groups (data were not shown). For 24 h reading, all groups showed a significant result, as compared with the negative control group. The data recorded for PMX205 in 24 h plate was in between 0.1311 to 0.1709, for EP54 was in between 0.09593 to 0.1357 and for group Tamoxifen was in the range of 0.1172 to 0.1570. While in 48 h plate, the value recorded for each significant group was determined. The readings for PMX205, the range was in between 0.1511 to 0.1623 while for EP54; the range was between 0.1282 to 0.1394 and for Tamoxifen, the range was between 0.1560 to 0.1671. For the last plate, which was 72 h plate, all groups PMX205, EP54 and Tamoxifen, once again showed a significant result as compared with control group. Each recorded at between 0.1280 to 0.1618, 0.1035 to 0.1373, and 0.1461 to 0.1799 respectively.
Figure 4: Alamar blue assay at 570 nm, wavelength. In 24 h, 48 h and 72 h time points, the result obtained were significant for each group when compared with the control group. Data for each bar is expressed as a mean value of absorbance reading for each type of groups. * indicating the significant values (P < 0.05), in which Dunnett post-hoc test is used in analyzing the data

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Figure 5: Percentage Reduction of Alamar Blue. The percentage reduction is calculated after correcting the overlap effect of the oxidized spectra at 600 nm wavelengths. Data for the line graph is expressed as a mean value of the percentage calculation for each time point. The line graph is represented to show the overall reduction of the Alamar blue after enzymatic transformation take place from time to time. * indicating the significant values (P < 0.05), in which Dunnett post-hoc test is used in analyzing the data

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Measuring Alamar blue absorbance at 570 nm shows statistically significant differences between each group when compared with negative control group. To reduce the overlapped effect of the oxidized spectra, the percentage of reduction was calculated and compared between each time point. The percentage reduction line graph showed a deflation data. Each time point showed an overall decreased value. For example, this is shown by a representative group EP54, where at 24 h, the data recorded was at 43.51% of reduction followed by 13.58% at 48 h and 18.62% at 72 h. This style of reduction was also been shown by the other two groups, PMX205 and Tamoxifen (data not shown).

MTT assay

In the MTT assay, [Figure 6] the data showed the presence of significant result in all time points. In 24 h plate, it showed that all groups were significant when compared with negative control group. For PMX205 group, the value recorded was in between 0.09893 to 0.2465, EP54 group, 0.02724 to 0.1748 and Tamoxifen group, the value recorded in between 0.09880 to 0.2464. For the 48 h plate of incubation time, only two groups showed a significant result, which were PMX205 and Tamoxifen. The values recorded were in between 0.04987 to 0.3273 and 0.5777 to 0.8551 respectively. For the 72 h plate, only one group showed a significant result, PMX205 (antagonist group) with the value recorded in between 0.02136 to 0.5322.
Figure 6: MTT assay results at 565 nm, wavelength. Results showed that at 24 h all groups obtained significant result when compared with negative control group. While in 48 h plate, it showed that there was a significant difference in PMX205, Tamoxifen with the control group while only PMX205 group showed significant result at 72 h time point. Data for each bar is expressed as a mean value of absorbance reading for each type of groups. * indicates the significant values (P < 0.05)

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 > Discussions Top


This experiment was conducted to detect the expression of C5a receptor on the 4T1 cell line, which is a malignant murine mammary cancer. Based on the result from the immunoflourescence staining, C5a receptor was expressed on the membrane of malignant murine mammary tumor, 4T1 cell line. This result was consistent with the PCR findings. Semi-quantitative PCR results showed that the C5a receptor gene was expressed in the cell by using 409 bp primer [18] while housekeeping gene primer GAPDH with 229 bp.[19] From semi-quantitative to quantitative, same RNA isolated was continued with RT-PCR to determine at which cycle the C5aR gene starts to be amplified. From qPCR, it showed that 4T1 cell line expressed the C5a receptor at cycle 22. Serial dilutions were done by using the RNA isolated and results obtained were increased over dilution factor (data not shown). Therefore, the higher the CT value obtained, the lower concentrations of C5aR were expressed on the cells.

To our knowledge, there are unearth reports recorded on the expression of the C5a receptor involving 4T1 cell line. There was only one report recorded on the expression of the C5aR on the benign murine mammary tumor, EMT6.[14] There were few reports recorded on determination of the expression of the C5a receptors on the individual target cell.[20],[21],[22] This is possibly because of C5a is a part of the large C5 protein in which structure and function depends on the specific secondary structure involving disulphide bonds and post-translational processing.[23]

The expression of complement C5a receptor, C5aR, has recently been demonstrated in several tumor types, including the syngeneic model of cervical cancer in mice [12] as well as a report on the expression of C5a receptor (CD88) mRNA in canine mammary tumors.[7] The C5a receptor, CD88, has also been expressed in a subset of normal bovine mammary epithelial cells.[24] One early report was recorded involving the complement activation on breast cancer cells in human tissue.[25] However, the expression of C5a receptor in cells may be terminated with the complement activation. However, the interaction occurred would need further investigations to know whether this interaction can bring up onto the formation or regression of the tumor itself. Tumorigenesis is also related with the occurrence of the inflammation at the site where tumor is developed. The role of inflammation in the tumorigenesis is still unclear as there are many contradicting theories that it promotes tumor growth or vice-versa. According to Coussens and Werb [26], the components of the immune system may act as a promoter to tumor development whereas according to Swann and Smyth [27], inflammation could protect against tumor development. The interaction between C5a and its receptor, CD88 is responsible for several kinds of diseases involving chronic inflammatory reactions [9],[10],[28] and over-activation of the major receptor for C5a, CD88, in cervical tissues has been hypothesized to promote tumor progression.[12]

The malignant mammary tumor, 4T1 cell line is continually investigated by using several types of drugs, which are agonist (EP54), antagonist (PMX205), and Tamoxifen as a positive control. Each of these drugs play a different function when it reacts with the cell line, 4T1. EP54 peptide, YSFKPMPLAR, is a potent, prominent agonist, which is being used as a functional probe to C5a receptor (C5aR) to activate complement system.[16] The activation of complement system eventually can promote the regression of the tumor itself. Different for PMX205, hydrocinnamate-OPdChaWR is an antagonist to the C5a receptor present on the cell itself that promotes the inactivation of the complement system in the murine model.[29] Tamoxifen is an anti-breast cancer drug, which is often being used for the treatment of advanced-stage estrogen receptor positive breast cancer.[30]

In this study, Alamar blue and MTT assays were used to study the effects of growth and viability of the 4T1 cell line through the treatment of the stated drugs. Generally, both of the assays had same objectives and rely on the enzymatic transformation.[31] However, previous report stated that Alamar blue is homogenous and has the advantage of higher sensitivity in terms of the detection of cell densities to as low as 200 cells/well.[32] Previous researcher had investigated on the comparison of Alamar blue and MTT assays for high throughput screening. The results suggested that both of the assays are of high quality to identify cytotoxic compounds without depending on the underlying mechanism and chemical classes.[33] This statement proved that both the assays are eligible to be used and able to determine the potential intrinsic cytotoxicity of the peptides and drug used in this study.

The results from the Alamar blue and MTT assays presented herein for the treatment of the cell line using three types of drugs were generally significant when compared to the negative control group. PMX205, which is an antagonist peptide, showed more effectiveness in treating the tumor as compared to the EP54 peptides. PMX205 was an inhibitor drugs that was used as an alternative to prevent the progression of tumor. Based on the results obtained, both of the assays showed consistency on the level of PMX205, which was lower as compared with EP54 treatment bar at each hour of incubation period. This result proved that repression of the binding of C5a to its receptors showed decreased inflammatory response. Thus, the reaction is not harmful to the defense system of the complement.[34] Once the receptor of C5a, CD88 is blocked, the complement system will not be fully activated. This is because of overexpression of the complement system that also can cause more inflammation to occur and thereby promoting cancerous cell to develop.[35]

The data obtained from the cell viability assays (Alamar Blue and MTT) showed the effectiveness of the antagonist peptide, PMX205, against the malignant mammary tumor cell. The results showed in this study may contribute towards the discovery of a new drug that can be used against cancer. Despite all the positive and significant results obtained from these experiments, further studies are clearly warranted to understand the effectiveness of the drugs used in the treatment of the tumor. It is also essential to understand the effects of C5a/CD88 itself on the tumor development and the response of immune system to it. The drugs might react differently in the host to defend against the tumor. Therefore, the use of rodent animal model as in vivo study is the best option to understand the mechanism of tumor regression following the activation of complement C5a protein.


 > Conclusions Top


This in vitro study provided the opportunity to study the effects of C5a on tumor without the confounding effects of immune cells or their products. These results provided the first evidence to support the possibility that C5a can act directly on the tumor cells themselves. However, these results should be treated with caution as they are from only one tumor type. Further studies are required to confirm these preliminary results, both in other cell types and by using other assays of biological activity.In vivo studies are also required to determine how these drugs would affect the tumor cell growth in the presence of a functioning immune system.


 > Acknowledgements Top


The authors would like to thank Dr. Trent M. Woodruff from School of Biomedical Sciences, University of Queensland, Australia for guidance and assistance in this project. This work is supported in part by a grant number, 02-01-04-SF1206 from the National Biotechnology Division, Ministry of Science, Technology and Innovation (MOSTI), Malaysia. Special thanks to members of Pharmacology/Toxicologic Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM) for their help and cooperation.

 
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