|Year : 2014 | Volume
| Issue : 1 | Page : 133-141
How prostate-specific membrane antigen level may be correlated with stemness in prostate cancer stem cell-like cell populations?
Toloudi Maria, Apostolou Panagiotis, Chatziioannou Marina, Kourtidou Eleni, Vlachou Ioanna, Mimikakou Georgia, Papasotiriou Ioannis
Research Genetic Cancer Centre Ltd, Megalou Alexandrou 115 Street, 53070, Filotas, Greece
|Date of Web Publication||23-Apr-2014|
Megalou Alexandrou 115 Street, 53070, Filotas
Source of Support: None, Conflict of Interest: None
Background: Prostate-specific membrane antigen (PSMA) is a widely used targeted molecule in prostate patients. The present research, attempts to support the hypothesis that PSMA expression in prostate cancer stem cell-like (CSC) cell populations may be correlated with nanog and other transcription factors in different stages of prostate carcinomas.
Materials and Methods: To provide more accurate evidence of the above, a population of prostate CSCs was isolated and analyzed using different protocols. The first method was based in the ability of CSCs to form spherical colonies in semi-suspension of a culture. A qPCRbased protocol and a flow cytometric analysis protocol were chosen to test the presence of stemness markers and PSMA in the selected populations.
Results: The formation of micro-sphere in semi-suspension has been pointed out. In the other panels of the test, the linear correlation between PSMA and nanog in gene and protein level was shown. However, the statistical analysis including the coefficient of variationand standard deviation's values) has proved that there were differences in PSMA expression between cancer cells and CSCs.
Conclusion: The previous analysis has pointed out that PSMA expression may be correlated with nanog's expression as well as with other confounders in a population of prostate CSCs.
Keywords: Cancer stem cell-like cells, prostate cancer, prostate-specific membrane antigen, stemness
|How to cite this article:|
Maria T, Panagiotis A, Marina C, Eleni K, Ioanna V, Georgia M, Ioannis P. How prostate-specific membrane antigen level may be correlated with stemness in prostate cancer stem cell-like cell populations?. J Can Res Ther 2014;10:133-41
|How to cite this URL:|
Maria T, Panagiotis A, Marina C, Eleni K, Ioanna V, Georgia M, Ioannis P. How prostate-specific membrane antigen level may be correlated with stemness in prostate cancer stem cell-like cell populations?. J Can Res Ther [serial online] 2014 [cited 2019 Sep 20];10:133-41. Available from: http://www.cancerjournal.net/text.asp?2014/10/1/133/131461
| > Introduction|| |
As prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males, the need for diagnosis, prognosis even treatment becomes crucial. Until now, many trials have been made to find a cure for this clinically important tumor but they were followed by some disadvantages because the age, the race (black), and the family history remain a well-established risk factor and it is difficult to prevent it. ,
However, in the last decades the hypothesis concerning the cancer stem cells (CSCs) became a cornerstone in the treatment of the disease.  Their role in the development of prostate cancer has been a research focus area for many years.
Many, if not all, tumors include a population of cells which have the ability to self-renew as well as to expand with high rates. This small population, known as CSC cells, may be a part of a population of circulating tumor cells (CTCs) that can be isolated from patient's blood sample. 
Previous studies have shown that CSCs are characterized by hallmarks such as surface and intracellular enzyme markers. Nanog gene is one of the most well-characterized stemness markers and in relation with oct3/4 (octamer-binding transcription factor 3/4) and sox 2 (also known as Sex determining region Y (SRY)-box 2) genes are a widely used triplet in describing a population of CSCs. During the epithelial-to-mesenchymal transition (EMT), cancer cells lose their epithelial differentiation and develop a mesenchymal phenotype. This is the reason that these cells acquire a sphere formation when cultivated in semi-suspension. ,,
Concerning the patients who suffer from prostate carcinoma, prostate-specific membrane antigen (PSMA), which is a membrane-bound glycoprotein has been widely used by many researchers and oncologists to combine the PSMA's expression level with a therapeutic model in the above group of patients. 
This study attempts to prove the hypothesis that PSMA expression may be correlated with nanog gene as well as with other confounders in a population of prostate CSCs.
| > Materials and Methods|| |
To test the association between PSMA level and prostate carcinoma, a series of different prostate cancers were analyzed successfully and different protocols were performed on prostate cancer cells as well as on prostate CSCs.
Peripheral blood was collected from 17 male patients who suffer from proven prostate carcinoma in different stages. To prevent coagulation, the sample was collected in tubes (Vacutainer K3E, 368860, BD (Becton, Dickinson and Company)) containing EDTA and it was rotated for about 30 min before use. All the samples were collected and analyzed with the consent of the patients.
Cancer cells were obtained from three human prostate cancer cell lines provided by the European Collection of Cell Cultures (ECACC). LNCap nuclear extracted (clone FGC) (89110211, Human Caucasian prostate carcinoma), 22Rv1 (05092802, Human Xenograft Prostate), and PC-3 (90112714, Human Caucasian prostate adenocarcinoma) were cultivated and used as controls.
To isolate CTCs from whole blood sample, Biocoll (L6115, Biochrom AG), a separating solution with density 1.077 g/ml, isotonic , was used. This solution (D = 1.077 at + 20°C) contains Ficoll400 (Polysucrose 400), a polymer with a molecular weight of approximately 400,000 Dalton. Biocoll separating solution was added in 15 ml centrifuge tubes (188271, Greiner Bio-One GmbH Maybachstr. 272636 Frickenhausen Germany Gmbh) and equal parts of whole blood were carefully applied on top of it. After a centrifugation step at 2500 rpm for 20 min, the layer of enriched (70-100%) lymphocytes between the plasma and Biocoll was isolated using a pasteur pipette. The collected cells were finally washed twice with phosphate buffer saline (PBS) (P3621, SIGMA-Aldrich, Life Science Chemilab S.A. Athens, Greece) and then divided and cultured in 25 cm 2 flask (5520100, Orange Scientific n.v./s.a., TECHNOLAB | Andreoiu Dimitriou 38, Athens, Greece) with Dulbecco's Modified Eagle Medium (DMEM) (D5546, SIGMA ?Aldrich?) as well as with STEMPRO hESC ?serum??and feeder?free medium (SFM) kit?(A10007?01, ??Life Technologies). Because cancer cells have infinite potential for division, those that remained in the flask after 1?week of culture were the cells of preference and normally a subset of them may had stemness phenotype.-Aldrich ) as well as with STEMPRO hESC serum- and feeder-free medium (SFM) kit (A10007-01, Life Technologies). Because cancer cells have infinite potential for division, those that remained in the flask after 1 week of culture were the cells of preference and normally a subset of them may had stemness phenotype. ,,
STEMPRO hESC SFM (serum- and feeder-free medium) kit provided the growth of human mammary epithelial cells and the highest quality of the microsphere culture. Itcomposed of DMEM/F-12 + GlutaMAX (1X) cultivation medium and the additional ingredients of FGF-basic factor (10 μg/ml, F029, SIGMA Aldrich), STEMPRO hESC SFM Growth Supplement (50X), 25% BSA (Bovine Serum Albumin), and 55 mM of 2-mercaptoethanol (M3148, SIGMA Aldrich).
So, the three commercial cell lines as well as the patient's cell lines, were cultivated both in the recommended culture medium with the appropriate amount of heat inactivated fetal bovine serum (FBS Superior; standardized BS, EU-approved, S0615, Biochrom AG) and 2 mM Glutamine (G5792, SIGMA Aldrich,) and in the STEMPRO product (complete medium) using 25 cm 2 flasks (5520100, Orange Scientific n.v./s.a.) at 37°C and 5% CO 2 atmosphere. Due to the fact that the environmental culture conditions play a critical role in the expansion, growth, and differentiation of cells, by cultivating the same starting cell population in different media, it would be possible to find differences between them concerning their genotype as well as their phenotype. All the experiments were performed during exponential phase and after 80-90% confluence of the culture.
The first scientific approach was an evaluation method which was based on the fact that CSCs, under special conditions, via epithelial-to-mesenchymal transition (EMT), lost their ability to detach and so they formed spheres and gained a mesenchymal phenotype. Prostate spheres are spheroid structures which can be passaged serially to generate daughter spheres with similar composition demonstrating that sphere-forming cells are capable of self-renewal. , By using a light microscope, the spheres could be observed in semi-suspension in a culture.
The second method used was the real-time Polymerase Chain Reaction (PCR) method which was the most sensitive, simple and quick method for studying the gene expression. It requires small amounts of template and thus it is a widely used research tool.
It has been proved that the population of CSCs is generally characterized by several molecular markers such as nanog, oct3/4, and sox2. Additionally, it was shown that prostate cancer cells expressed PSMA in different tumor stages. Concerning this approach, RNA was extracted using Trizol reagent (15596-026, Life Technologies) and used as template to synthesize cDNA with the use of the First Strand cDNA Synthesis (K1612, Fermentas, Thermo Fisher Scientific Inc). To test gene's expression, a quantitative real-time PCR protocol was run (K0221, Maxima Sybr Green, Fermentas, Thermo Fisher Scientific Inc) using as endogenous control the 18S rRNA gene [Table 1].
The primers used were designed using Genamics expression program (Genamics Expression DNA Sequence Analysis Software, version 1.100≥ 2000) [Table 1]. All the designed sequences were run on BLAST to exclude those who amplified undesired genes. The real-time PCR protocol included a denaturation program (94°C for 10 min), an amplification, and quantification program repeated 50 times (94°C for 15 s, 59°C for 15 s, and 72°C for 30 s) with a single fluorescent measurement, a melting curve program (55 -95°C with a heating rate of 0.5°C/s and a continuous fluorescent measurement), and finally a cooling step at 4°C. Each sample was amplified in triplicates. At the end of the reaction the detection of the cycle-threshold (which is the level of detection of the point at which a reaction reaches a fluorescent intensity above background) was made. Finally, the genes' relative expression was calculated using the 2−ΔΔCT (Livak) method. 
To determine the expression of the above-mentioned genes in a protein level, flow cytometric analysis was performed (BD Accuri™ C6 Flow Cytometer, BD Biosciences) . ,,,, The antibodies that were used to evaluate the expression levels of them in the test population was a nanog mouse anti-human antibody conjugated with phycoerythrin (PE, 560483, BD Biosciences ) as well as an anti-PSMA antibody conjugated with PE (K0142-5, MBL International) as well. For the data analysis, the FCS Express Flow Cytometry Data Analysis De Novo Software (FCS Express V3, version 3.00.0504, Professional Stadalone, 2001-2007) was used.
| > Results|| |
Concerning the sphere-formation evaluation assay, the ability of CSCs to form spherical colonies under enabling conditions was pointed out. When the three selected commercial human prostate cancer cell lines ( LNCAP, 22Rv1, and PC-3) were cultivated in two different culture media (the first was the recommended growth medium for each cell line and the second was the STEMPRO hESC serum- and feeder-free medium (SFM) kit, which providedthe growth of human mammary epithelial cells and the highest quality of the microshere culture, as it was mentioned before), spherical colonies were observed in the supernatant of STEMPRO- cultivated cells only [Figure 1],[Figure 2],[Figure 3] and [Figure 4]. The same phenotype was defined in prostate CSC cell lines when they were cultivated in DMEM and STEMPRO medium, respectively [Figure 5] and [Figure 6].
|Figure 1: 22Rv1 human xenograft prostate cancer cells were cultivated in RPMI without phenol red medium (R7509, SIGMA Aldrich). The cells, as it was expected, formed a matrix and they were detached to the flask|
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|Figure 2: 22Rv1 human xenograft prostate cancer cells were cultivated in STEMPRO hESC SFM (serum- and feeder-free medium) kit (A10007-01, Life Technologies). Obviously, the cells started to form spheres to semi-suspension|
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|Figure 3: LNCap nuclear extracted (clone FGC) human caucasian prostate cancer cells were cultivated in RPMI 1640 medium (R0883, SIGMA Aldrich). The cells normally formed a matrix and they were detached to the flask|
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|Figure 4: LNCap nuclear extracted (clone FGC) human caucasian prostate cancer cells were cultivated in STEMPRO hESC SFM (serum- and feeder-free medium) kit (A10007-01, Life Technologies). This figure also represents cells of the same starting population which were cultivated with different culture mediums and they did not form a matrix. On the contrary, this population started forming spheres to semi-suspension|
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|Figure 5: Prostate cancer stem cell-like cells were isolated from a prostate patient's blood sample (stage IV) and were cultivated in STEMPRO hESC SFM (serum- and feeder-free medium) kit (A10007- 01, Life Technologies).. The sphere formation is represented here|
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|Figure 6: Prostate cancer stem cell-like cells were isolated from different prostate patient's blood sample (Gleason rate 7) and were cultivated in STEMPRO hESC SFM (serum- and feeder-free medium) kit (A10007- 01, Life Technologies). The sphere formation is represented here|
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According to the qPCR data analysis that wasperformed using relative quantification (normalized to the endogenous gene, 18S rRNA), on all patients who were selected and analyzed, it was observed that the three stemness markers (nanog, oct3/4, sox2) were expressed in higher levels in STEMPRO-cultivated cells than those in DMEM medium. Indicatively, in a prostate CSC population (which was isolated from a prostate patient with stage IV) the expression of nanog in the STEMPRO-cultivated cells was 1.64 times higher than its expression in DMEM-cultivated cells (using as the 18S rRNA gene as reference gene). A similar pattern was observed for the other stemness markers for the same population [Table 2]. It has also been shown that PSMA's relative expression is increased when it is correlated with the relative expression of nanog in a prostate CSC population that was isolated form a prostate patient, stage Ic in comparison with cancer cells (in DMEM medium) (using the Livak method) [Table 3]. Additionally, it was pointed out that the correlation between PSMA and nanog was linear (because their relation is described by the equation y = αx + b) as well as really high (because of the R2 measurements). Comparing the above statistic results in the two different culturing conditions, it was observed that the mentioned correlation between PSMA and nanog was higher in DMEM than in STEMPRO-cultivated cells [Figure 7].
|Figure 7: Linear correlation between prostate-specific membrane antigen and nanog gene. Although the correlation between the two selected genes was high in both diagrams (R2 = 0.875 and R2 = 0.8745, respectively), the coefficient of variation in the second diagram (concerning STEMPRO-cultivated cells) was higher than in the first which means that the gene expression of prostate-specific membrane antigen in the prostate cancer stem cell-like cells population may be induced by other confounders (coefficient of variation, 0.382 <0.715, respectively)|
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|Table 2: Analysis of relative gene expression data using real-time quantitative PCR and the 2−ÄÄCt Method concerning three stemness markers|
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|Table 3: Analysis of relative gene expression data using real-time quantitative PCR and the 2−ÄÄCt Method concerning nanog and PSMA gene|
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To check the protein levels of the selected stemness triplicate, a flow cytometric-based assay was used. The PSMA protein expression was confirmed in the three commercial human prostate cancer cell lines (22Rv1, LNCaP, and PC-3 cell line). As it is mentioned by other researches the protein expression levels were higher in 22Rv1 and LNCaP cell lines than in PC-3 [Figure 8],[Figure 9] and [Figure 10].
|Figure 8: Prostate-specific membrane antigen protein level in 22Rv1 human xenograft prostate cancer cells which were cultivated in RPMI without phenol red medium. The percentage is 40.74%|
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|Figure 9: Prostate-specific membrane antigen protein level in LNCap (clone FGC) human caucasian prostate cancer cells which were cultivated in RPMI 1640 medium with 1 mM Hepes and 1 mM sodium pyruvate. The percentage is 54.76%|
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|Figure 10: Prostate-specifi c membrane antigen protein level in PC3 human Caucasian prostate adenocarcinoma cells which were cultivated in AQ2 DMEM medium. The percentage is very low (0.83%)|
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Concerning the protein levels of PSMA and nanog, it is crucial to refer that according to the statistical analysis that has been performed, the correlation between them remained in high level as it also was found concerning their gene expression. But in this case the difference between the two different culturing populations tended to be much more critical [Figure 11].
|Figure 11: Linear correlation between prostate-specific membrane antigen and nanog protein. The correlation between the two selected proteins was high in both diagrams (R2 = 0.9833 and R2 = 0.9639, respectively) but the coefficient of variation in the second diagram (concerning STEMPRO -cultivated cells) was much higher than in the first which means that the expression of prostate-specific membrane antigen protein in the prostate cancer stem cell-like cells population may be induced by other confounders (coefficient of variation, 0.566 < 11.623, respectively)|
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| > Discussion|| |
The prostate cancer is a form of malignancy that is developed in the prostate gland. The rates of disease detection vary widely across the world with the most frequent detection in Europe.
Most prostate cancers are growing slowly; however, there are cases of aggressive prostate cancers. A better scientific approach is needed to understand the triggering factor (s) that influence the genetic and biologic mechanisms of CSCs and the dormant cell population becomes active.
Many experiments were performed by the scientific community to elucidate the possible role of serial markers including PSMA, Prostate-specific Antigen (PSA), human kallikrein type 2, Bone Alkaline Phosphatase, Total Alkaline Phosphataseetc. in tumor progression. A well-characterized experimental as well as therapeutic model is the duality PSMA-PSA. ,
PSMA is a type II membrane glycoprotein with folate hydrolase activity produced by prostatic epithelium. The expression of this molecule has also been documented in extra-prostatic tissues, including small bowel and brain. It is a well-known imaging biomarker for staging and monitoring therapy. Because it is highly restricted to the prostate and overexpressed in all tumor stages, it represents an attractive antigen for antibody-based diagnostic and therapeutic intervention in prostate cancer. , On the other hand, PSAis also a protein produced by normal prostate cells. This enzyme participates in the dissolution of the seminal fluid coagulum and plays an important role in fertility. The highest amounts of PSA are found in the seminal fluid. When a low amount escapes the prostate thus can be found in the serum, it is said that rising levels of PSA in serum are associated with prostate cancer. 
According to previous findings, there were various profiles of PSMA and/or PSA expression levels in different prostatic groups. It has been shown that when the PSMA-/PSA + profile decreased in normal prostates, benign prostatic tissue, and primary prostate cancers, the PSMA + /PSA- level increased from normal to prostate tumor tissues. Generally, researchers support the idea that the amount of PSMA and/or PSA in healthy men is low in the blood and it increases either with age or because of inflammation of prostate gland (prostatitis) or prostatecancer ,,,,
To decrease the side effects of any prostate therapy, it is crucial to develop a good screening pattern for preventing the disease. Finding a tumor at an early stage may be easier to treat before it will cause serious symptoms. For that reason, a small population of cells may be a key in tumor initiation and progression. This small population, which was identified and characterized in some tumor types a few years ago, is called CSCs. Basic hallmarks of them are self-renewal and the ability to differentiate into multiple cell types. The CSC's hypothesis indicates that targeting in this population may be the cause of preventing tumor metastasis and relapses ,,
In our study, prostate CSCs populations were collected from patients who suffered from prostate carcinomas and were cultivated for analysis. It has been found that under special conditions the prostate CSCs started forming spheres in semi-suspension. A mesenchymal phenotype was developed instead of their epithelial characteristics that they had before the EMT. This formation, which was observed in all STEMPRO cultures of the analyzed prostate patients, is a powerful initial research tool of identifying CSCs in a culture.
On the other hand, the expression of a gene triplet (nanog, oct3/4, and sox2) was tested in comparison with PSMA expression, using real-time PCR protocols and flow cytometric analysis. From the data analysis, the great correlation between PSMA and nanog either in gene or in protein level has been shown. The straight line in the graphs described the linearity between them. Finding that the coefficient of variation in DMEM cultures was much higher than in STEMPRO cultures indicated that some other confounders may play a crucial role in the expression of PSMA in these populations of prostate CSCs. It would be helpful to search which other genes such as prostate stem cell antigen or molecular pathways are implicated in prostate cancer development and metastasis.
| > Conclusion|| |
has gained increased attention in diagnosis, monitoring, and treatment. Undoubtedly, it represents a good prognostic marker in prostate carcinomas. From all the previous analysis, it has been shown that the presence of PSMA in a population of prostate CSCs may be a key in preventing and/or diagnose of the disease. But still, extended experiments need to be performed to prove that the correlation does not imply causation and the PSMA expression may be induced by other additional crucial factors.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2], [Table 3]