P-element induced wimpy testis-like 2 (Piwil2) is in the Piwi gene family. Piwil2 has important roles in the self-renewal mechanism of stem cell induction and progression of numerous types of human malignancies such as lung, breast, colon, prostate, and cervical cancers. Glutathione
MCF-7/Piwil2 and MCF10A/Piwil2, transfected with a plasmid carrying the Piwil2 gene, and non-transfected MCF-7 and MCF10A were cultured in a complete DMEM/F12 medium. GST A1 and P1 activity was determined in these cell lines using as substrates CDNB, EA respectively.
According to experimental results, GST P1 activity decreased in the MCF-7/Piwil2 cells as compared with the non-transfected MCF-7 cells, however, MCF-7/Piwil2 cells demonstrated increases in GST A1 (total GST) activity. The statistically significant differences were found for the comparison of non-transfected MCF-7 and MCF-7/Piwil2 (p<0,0001), for GST enzyme activities by using CDNB and EA as substrates. These results were the same for the MCF10A cell line.
It is shown for the first time that transfection studies may affect GST activity at the cellular mechanism level. The study contributes to determining the effect of transfection on GST isoenzymes and also how the Piwil2 gene may affect GST activity in the stem cell line.
- cancer stem cell
- ethacrynic acid
- glutathione -transferase
In eukaryotic and prokaryotic organisms, GSTs are divided into cytosolic GST, mitochondrial GST, and microsomal GSTs . GST isoenzymes synthesized from human tissues consist of Alpha (α), Mu (µ), Pi (π), Sigma (σ), Theta (θ), Kappa (κ), Omega (ω) and Zeta (ζ) gene families. Each of the gene families’ GSTs differ in substrate and inhibitor specificity patterns. Cytosolic GST consists of a large number of protein subunits. These proteins are encoded by different genes: GST a (GST A1, A2, A3, A4, GST 9.9 and GST x), GST l (GST M1, M2, M3, M4, and M5), GST p (GST P1), GST h (GST T1 and T2) . All GST classes are structurally different from each other. However, the common feature of these GST classes is to reduce the pKa of the GSH’s tiol group from 9.0 to 6.0 to 6.9. Thus, it strengthens the nucleophilic attack of GSH against electrophilic co-substrates .
GSTs metabolize the side effects of cancer chemotherapy drugs, insecticides, herbicides, and by-products of oxidative stress. They are also of interest to pharmacologists and toxicologists as they provide targets for anti-asthma and anti-tumour drug therapies. Excessive GST activity in human cancer cells has been associated with the ability to fight chemotherapy drugs and carcinogens .
“Piwil2 is in the Piwi gene family, whose members are defined by maintained Piwi and PAZ domains, and it has important roles in the self-renewal mechanism of stem cells, RNA silencing and translational regulation in diverse organisms” . The Argonaute family’s eight members are identified as human. This family is classified into two subfamilies, the Piwi subfamily: Piwil1 (hiwi), Piwil2 (hili), Piwil3 and Piwil4 (hiwi2), and the eIF2C/AGO subfamily. Members of the Piwi gene family, both mouse and human Piwil2 genes, are expressed specifically in the testis and play an important role in spermatogenesis. Piwil2 gene was understood to be expressed in other kinds of cancer but was considered a new member of testicular antigens and as an apoptosis inhibitor as well [4, 5]. However, it has an important role in tumor induction, proliferation, and survival . Piwil2 is thought to be a cellular marker for precancerous stem cells (pCSCs), and the regulation of carcinogenesis [6, 7, 8]. Alternative mRNA peptides of the Piwil2 gene have been found in pCSCs. One of these peptides, P12L60 can activate carcinogenesis in the lack of the protein coded with the Piwil2 gene .
There are a number of studies involving many different methods done on the GST and Piwil2 gene using cancer cell lines separately, however no study has examined the GSTs level of the effect of Piwil2 expression on cancer stem cells so far. Determining how GST levels are affected as a result of Piwil2 expression is very important in understanding cancer and drug resistance metabolism. From this point of view, this type of study could provide the opportunity for the initiation and development of a human tumor in vitro.
This study aimed to investigate the effect of Piwil2 expression on these cell lines by determining the activity and levels of metabolic proteins at the molecular level, after the transfer of stem cell protein Piwil2 to human breast epithelial cells and human breast cancer stem cells.
The human breast adenocarcinoma cell line, Michigan Cancer Foundation-7 (MCF-7), was obtained from Dr. Gökçen Dinç; MCF10A, a non-tumorigenic epithelial cell line, and Piwil2 were provided by Dr. Servet Özcan, from the Genome and Stem Cell Center, Kayseri, Turkey. MCF-7 and MCF10A cells were transfected with a plasmid carrying the Piwil2 gene, in accordance with the manufacturer’s instructions. Non-transfected MCF-7 and MCF-7/Piwil2 cells were cultured in a DMEM/F12 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 µg/ml sodium pyruvate, 0.01 mg/ml human insulin and 1% penicillin/streptomycin, maintained at 37°C in 5% CO2. MCF10A and MCF10A/Piwil2 (control groups) cells were cultured in a DMEM/F12 medium containing 20% fetal bovine serum, 2 mM glutamine and 1% penicillin/streptomycin, maintained at 37°C in 5% CO2 for 2 days without any application. For the selection of the transfected cells, G418 was applied at 400 µg/mL for two weeks at the medium of two days intervals. After the selection procedure, adherent wild type MCF-7, MCF10A, and transfected cells with Piwil2 were trypsinized and the cell suspension was centrifuged at 1800 × g for 5 min at 4°C. Pellet was resuspended in homogenization buffer (25 mM potassium phosphate, pH:6.5, 1 mM EDTA), whereupon the cell suspension was sonicated for 10-second periods 5 times, before being centrifuged at 15000 × g for 10 min at 4°C. The supernatant was used for the determination of protein by the Bradford method  and enzyme activities.
GST activity was measured with the Habig optimized method . Total GST activity, GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) (ε = 9.6 mM−1 cm−1), etacrynic acid (EA) (ε = 5.0 mM−1 cm−1) were used as substrates for total GST, GST Pi, activity determinations at 340 nm, 270 nm, respectively. Activity determinations were performed with comprised reduced glutathione (GSH) concentrations of 1 mM CDNB with 1mM GSH (1x PBS tampon pH 6.9, 43 mM Na2HPO4, 14.7 mM KH2PO4, 0.137 mM NaCl, 2.7 mM KCl) 0.2 mM EA with 0.25 mM (0.1 M potassium phosphate tampon, pH 6.5) in a 1000 µl assay reaction mixture. During the experiments, the continuous absorbance readings were obtained from the same sample at 5-minute intervals by spectrophotometer 9 times to evaluate statistical result sensitivity.
Non-transfected MCF10A, MCF10A/Piwil2, non-transfected MCF-7 and MCF-7/Piwil2 cells were seeded on coverslips in six plates, at a density of 3×105 cells/well in DMEM-F12 and incubated in a humidified CO2 incubator (5% CO2, 37°C). After 24 hours, cells were washed three times with PBS, were fixed with 4% paraformaldehyde in PBS for 10 minutes, and washed with PBS. Nonspecific binding sites were blocked with bovine serum albumin (IgG free BSA) by standing in 5% BSA/PBS solution for 1 hour at room temperature. Cells were then incubated overnight at 4ºC with primary antibody for MCF10A, MCF10A/Piwil2, non-transfected MCF-7 and MCF-7/Piwil2 separately. The primary antibodies were used at a dilution of 1:100 in 1% BSA/PBS and then washed with PBS, 5 minutes each time, then further incubated for two hours in the dark at room temperature, with a secondary antibody. The secondary antibody solution was diluted at 1:250 with 1% BSA/PBS for each of the cells followed by a wash with PBS, 5 minutes each time. Finally, cells were mounted with an antifade solution for viewing and photographing on a Nikon-Eclipse Ti-S fluorescence microscope applied to a Nis-elements 20x objective.
GraphPad Prism 6 software was used for statistical analysis. A two-tailed, unpaired Student’s t-test was used for significance analysis. A p-value of <.05 was accepted as statistically significant.
MCF10A cells appeared as elongated filamentous structures (Fig. 1a) and MCF-7 cells showed their multiple and triangular spikes (Fig. 1b). The flasks with the cells were controlled at the end of the third day to determine the presence of growth. At the end of the sixth day, for both cell lines, the plate-based cell density was observed to be 80–90% in the re-examined flasks.
In this study, the goal was to determine GST isoenzyme profiles in breast cancer cell lines transfected with Piwil2. Enzymatic activities for the control groups of non-transfected MCF10A and MCF10A/Piwil2, total GST mean (GST A1) activities by using CDNB as substrate, were found at 32,07±3,6 nmole−1min−1mg and 94,7±3,15 nmole−1min−1mg, respectively (Fig. 2a). For the enzymatic activities, total GST means activities were found to be 82,6±2,9 nmole−1min−1mg and 161,2±3,2 nmole−1min−1mg for MCF-7/WT and MC-7/Piwil2, using CDNB as substrate, respectively (Fig. 2b).
Enzymatic activities for the control group of non-transfected MCF10A and MCF10A/Piwil2, GST Pi mean (GST P1) activities, using EA as substrate, were found to be 52,8±5,7 nmole−1min−1mg and 34,9±2,5 nmole−1min−1mg, respectively (Fig. 3-a). GST Pi mean activities were found to be 143,5±3,4 nmole−1min−1mg and 52,5±3,5 nmole−1min−1mg for the control group of non-transfected MCF-7 and MCF-7/Piwil2, using EA as substrate, respectively (Fig. 3b).
Expression of Piwil2 in breast cell lines was examined by immunostaining analysis using an anti-Piwil2 antibody. The Piwil2 expression was observed in the cytoplasm of human breast cancer cell line MCF-7 and the non-tumorigenic epithelial cell line MCF10A (FITC). Non-transfected MCF10A (Fig. 4a) and MCF/7WT (Fig. 4b) had typical morphology. MCF10A (Fig. 5a) and MCF-7 cells (Fig. 5b), transfected with Piwil2, lost their filamentous and pointed multiple structures, showing a rounded and less spindled structure. It has been observed that Piwil2 transfection was expressed by almost 50% on both cells. The Piwil2 transfected MCF-7 were shown to over-express Piwil2 compared with Piwil2-transfected MCF10A cells and non-transfected cells.
In the study, p<0.0001 was considered statistically significant and the difference between the groups was found to be significant. The statistically significant differences were found for the comparison of non-transfected MCF-7 and MCF-7/Piwil2 p<0.0001 by using CDNB as the substrate for total GST activities. In addition, statistically significant differences were found for the comparison of non-transfected MCF-7 and MCF-7/Piwil2 p<0.0001 for GST P1 activities, using EA as a substrate. There was also a significant difference for the control groups.
Breast cancer has been investigated in laboratory and clinical settings for many years. However, the incidence of breast cancer is increasing. Piwil2 has important roles in the self-renewal mechanism of stem cell induction and progression of numerous types of human malignancies such as lung, breast, colon, prostate and cervical cancers. Therefore, tumor formation occurs due to disruptions in the expression of Piwi protein. Glutathione transferases were chosen because they are linked to other tumor traces, such as estrogen receptor proteins. In this study, the effect of Piwil2 expression on both tumor cells and non-tumor cells at the metabolic protein level was demonstrated for the first time applying the in vitro method. These findings extend observations of the differences in protein activity levels of GST isoenzymes that had been examined before and after Piwil2 transfection. In the human genome, several genes are represented by unique models in each tissue in class-specific clusters of different chromosomes. Most of these genes are polymorphic and cause variations of the GST phenotype. The genes encoding GSTT1, GSTM1, and GSTP1 are the main GST genes . Dong et al. have reported GSTs protecting the cell against oxidative stress and toxic substances. GST P1 is a phase II metabolic enzyme that facilitates cellular detoxification; it catalyzes the glutathione substrate conjugation to protect the macromolecule from oxidative stress. Thus, it protects cells against various toxic compounds. Excessive activity of GST P1 has been associated with the emergence of tumor resistance to antitumor drugs, indicating that this enzyme has functions beyond metabolism. This disclosure raises exciting possibilities in the field of drug discovery, as GST P1 inhibitors and their prodrugs offer a viable strategy for designing anticancer drugs whose primary purpose is to reverse tumor resistance . Mahajan et al. have reported that GST inhibitors can increase the sensitivity of cancer cells to anticancer compounds and therefore can be used in some therapeutic applications . In a wide variety of cancers, GSTs play a role in resistance to several chemotherapy drugs by their conjugating activity. GSTs generally show higher activity levels in cancer cells compared to normal cells [15, 16]. Punganuru et al. reported that EA is a GST P1 and other GSTs inhibitor with good pharmacological properties. Its inhibitory effect on GST P1 is active against human tumor cells. Punganuru et al. studied the drug feature concerning diuretic activity of ethacrynic acid-glucosamine conjugate (EAG) and EA on female CD-1 mice and demonstrated increments in diuretic activity of EA according to EAG. In human cancer cell lines, lung cancer cell line (H129), brain cancer cell lines (GBM10 and SF188), breast cancer cell lines (SKBR-3, MDAMB-231, MDA-MB-468, MCF-7), colon cancer cell lines (HT29 and HCT116), Punganuru’s group demonstrated an enhancement in cell growth inhibitory effects of EA levels on these cancer cell lines . Guneidy et al. examined protein levels of GST by using a CDNB substrate on breast cancer cell lines. Their comparison of increased GST A1 activities were found to have much similarity with our study . Aybek et al. investigated the effects of chemotherapeutic agents tamoxifen and 5-FU on MCF-7 cell line and human erythrocyte GST, an important enzyme of intracellular antioxidant metabolism. Their results showed that 5-FU and tamoxifen increased the total activity of GST . Zeng et al. investigated microsomal GST1 (MGST1) in lung adenocarcinoma cell lines (SPC-A-1, H2342, A549, AAH1975, and PC-9). They reported that MGST1 plays specific roles in oxidative stress, cancer occurrence, and multidrug resistance; it also plays a vital role in lung adenocarcinoma tumorigenesis and could serve as a potential prognostic factor in lung adenocarcinoma . Sanaei et al. examined the effect of 5-AZA-Cr epi-drug on GSTP1 at the gene level in the hepatocellular LCL-PI 11 cell line. They demonstrated enhancement of GST P1 gene expression by using the drug in hepatocellular cell lines . Considering all of these examples in the literature, it was reported that total GST activity decreased in control groups (non-tumorigenic cell line, serum, tissue), increased in cancer cell lines, and increased even more after treatment. It was stated that GST P1 activity was high in the control groups; this activity decreased in the cancer cell line but increased with treatment. According to this literature, where GST P1 activity was evaluated, this activity has increased in the control groups, decreased in the cancer cell line, but increased with treatment. However, a confirmation was also observed by showing higher GST A1 protein activity of the GST isoenzyme in non-tumorogenic cells after Piwil2 transfection, and also increasing GST A1 and decreasing GST P1 levels after Piwil2 transfection in the cancer cell line. These results of pre- and post-transfection with Piwil2 were confirmed by immunostaining for increase in GST A1 and the decrease in GST P1 results of transfection of Piwil2.
Piwil2 plays a regulatory role in the self-renewal of stem cells and tumorigenesis . Zhang et al. investigated the effect of Piwil2 on fibroblast by protein expression (matrix metalloproteinase [MMP] 2, MMP9, α smooth muscle actin (α-SMA), and vimentin and fibroblast-activating protein [FAP]). They isolated exosomes from Piwil2-induced cancer stem cells (Piwil2-iCSCs) and fibroblasts (FBs) served as recipient cells. They reported that exosomes induce cancer-related phenotypes in fibroblasts in vitro and CSCs can provide tumor growth by modulation of the tumor microenvironment . Feng et al. demonstrated that the β-catenin/CREB binding protein-mediated transcription activated by Piwil2 is essential for the contribution to the progression of cervical oncogenesis . Zou et al. demonstrated that the activation of Piwil2 triggered the alteration of purine metabolism and initiated the repair of lung pyrosis .
In this study, GST activity increased after the Piwil2 transfection using CDNB as a substrate and decreased after the Piwil2 transfection using EA as a substrate. It should be noted that the presence of numerical values, metabolic protein levels for drug discovery, and the Piwil2 gene, may affect GST expression in the human breast cell line, MCF-7.
This study investigated the effect of Piwil2 expression on both tumor cells and non-tumor cells at the metabolic protein level, using the in vitro method for the first time. After the Piwil2 transfection into MCF10A and MCF7, Piwil2 has increased cancerization at the cellular level in these cells. I presume that it could be helpful to contribute to the understanding of the function of the Piwil2 gene in the cell and its functioning at the molecular level, and also in determining breast cancer metabolism in GST activities.
The investigation demonstrated Piwil2 as a key player in the process of tumor initiation, progression, and invasion, in addition to suggesting that this factor can be used as a common cancer biomarker, as well as being used as an indicator in some significant transfections studies.