Abstract
In present study, it was purposed to determine the in vitro effect of the extract obtained from the pomegranate (Punica granatum L.) peel on the breast cancer cell line. MDA-MB-231 cells were exposed to pomegranate peel extract (PoPx) at 37 °C and 5% CO2 for varying durations (24 and 48 h) and doses (25 and 50 μg/mL). At the end of the incubation periods, argyrophilic nucleolus organizer regions (AgNOR) protein status, cell viability/apoptosis and cell cycle of MDA-MB-231 cells were examined in the Muse Cell Analyzer device. Cell viability was observed to be decreased when the groups treated with PoPx were compared with the control group. The group in which apoptosis was observed with the highest value was 50 μg/mL PoPx group (p < 0.05). In the cell cycle test, the number of cells in the G0/G1 stage was found to be significantly higher in the 25 μg/mL PoPx group compared to the control and 50 μg/mL PoPx groups at the end of the 24-h incubation period (p < 0.05) The results also supported cell cycle and apoptosis, and at the end of 24 h, Total AgNOR area(TAA)/Total nuclear area (NA) ratio and AgNOR numbered decreased on the 50 μg/mL PoPx group and were found to be statistically significant compared to the control group (p < 0.05). Consequently, it was determined that PoPx increased apoptosis on breast cancer cells by various mechanisms and inhibited cell viability/cell growth. This study showed that the widespread consumption of PoPx may be effective in preventing cancer formation and slowing its progression.
Keywords: MDA-MB-231, Punica granatum L, AgNOR staining
Graphical Abstract
Graphical Abstract.
Introduction
Cancer is a chronic disease that ranks second among the causes of death in the world and in our country.1 Cancer, which is a major public health problem,2 arises as a result of uncontrolled division and proliferation of cells. There are more than 100 types of cancer.3 Chemotherapy, radiotherapy and surgery are known as cancer treatment methods.4 In addition to these treatments, complementary and alternative treatment methods have been used recently.5 Nutrients that have positive effects on human health, play a role in preventing possible diseases and slowing the progression of chronic diseases are called functional nutrients.6 Pomegranate fruit is considered a functional food because of its phenolic compounds displaying antimicrobial, antitumoral, anti-inflammatory and antioxidant activities. Studies have reported that the antioxidant capacity of the pomegranate peel is two times higher than the seeds, and approximately 10 times higher than the edible fleshy part of the fruit.7
Nucleolus organizer regions (NOR) are regions of the ribosomal gene in chromosomes. These regions are composed of ribosomal DNA (rDNA) and some proteins with argyrophilic properties. These regions are replicated to the ribosomal RNA, which is transformed into the immature ribosomes in the nucleolus and mature ribosomes in the cytoplasm, respectively.8 These regions can be dyed with silver, when active. Depending on silver, these proteins are called argyrophilic NOR (AgNOR) concerned proteins, and the silver staining method is the most suitable to indicate nucleoli in the interphase nuclei.9,10 There are a few studies on the importance of interphase AgNOR quantification in tumor pathology for prognostic and diagnostic definitions of different types of cancer.11 In the literature review, no researches about the association between AgNOR proteins amount and the effects of PoPx treatment have been performed on MDA-MB-231. Therefore, we showed the present study to indicate any possible effects of pomegranate (P. granatum L.) treatment on the NOR protein synthesis and the selection of the most accurate dose for cancer treatment.
Materials and methods
Preparation of PoPx
The pomegranates (P. granatum L.) used in the study were obtained from a market in Kayseri province in Turkey. Pomegranate peels were manually separated from the fruit. The peels were left to dry in the shade with air circulation. After removing bruised and sunburnt peels, the dried peels were powdered with a blender (Waring, Staufen, Germany) and passed through a sieve to obtain uniform particles. Fifty grams of peel powder was mixed with 250 mL of methanol, and the mixture was placed into a magnetic stirrer (Daihan, Korea). Extraction was performed at room temperature with constant stirring at 250 rpm for 30 min. The resulting mixture was centrifuged (Hettich, Tutlingen, Germany) at 5,000 rpm for 5 min, and passed through a 0.45 μm PTFE filter. The solvent of the extract was evaporated at 40 °C with a rotary evaporator (Heidolph, Germany).
Analysis of phenolic compounds by HPLC
A slightly modified high performance liquid chromatography (HPLC) method of Çam and Hışıl12 was used for the determination of phenolic substances. PoPx was diluted 10 times and passed through a 0.45 μm PTFE filter, and injected into the HPLC system (Shimadzu, Kyoto, Japan). Separations were done by a Licrospher C18 column (250 × 4.6 mm, 5 μm). A binary gradient system was used with a flow rate of 1 mL/min. Mobile phase A was water acidified with trifluoroacetic acid (0.05%), and mobile phase B was a mixture of water-acetonitrile (3:7, v/v) acidified with trifluoroacetic acid (0.045%). The elution program was: 0% to 50% B from 0 to 15 min, 50% to 100% B from 15 to 17.5 min, 100% B to 20 min, and back to starting conditions in 5 min. The chromatogram was monitored at 378 nm with a PDA detector. Quantitation was based on an external standard method.
Cell culture
MDA-MB-231 cell line was derived from American Type Culture Collection (Manassas, VA, USA). The MDA-MB-231cells were cultured Dulbecco’s modified Eagles medium (DMEM Capricorn Scientific, CP21-4310) including streptomycin/penicillin (100 U/mL; Sigma Life Science, 046M4846V) and 15% fetal calf serum (FCS) (Biowest, S181G-500) in a humidified (Sanyo, MCO-19 A/C(UV)) atmosphere of 5% CO2 air at 37 °C. Sterile conditions were applied to avoid the risk of contamination. Then healthy MDA-MB-231cells were divided groups for PoPx treatment. Tissue culture plate with 24 well including 1,000 μL of medium with 1× 105 MDA-MB-231 cells was used to find optimum PoPx dose. These MDA-MB-231 cells was cultured was in a humidified atmosphere 95% air containing 5% CO2 overnight to attach to the plate. After the medium was removed and MDA-MB-231 cells were rinsed with 500 μL phosphate-buffered saline (PBS) 3 times. The MDA-MB-231 cells were identified. The experimental groups were generated as log concentrations of PoPx (25 and 50 μg/mL) on breast cancer cells after the different cultured period.
Cell viability assay and proliferation
The number of cells per ml of cell suspension was determined with Trypan Blue cell counting method. For cell counting, an amount of the cell suspension was taken into the Eppendorf tube and Trypan Blue solution was added in the same proportion as the amount taken from the cell. After 5 min of incubation, the coverslip was transferred to both sides of the closed Thoma slide (Marienfeld-Superior). Stained and unstained cells were noted by counting with a microscope (Nikon Eclipse TS100). The number of cells within the counting area was averaged. Number of viable cells (cells/mL) = Average number of cells × 2 × 104 was calculated according to the formula.
Experimental design
Annexin V and cell cycle assay for 24 and 48 h incubations and control, 25 and 50 μg/mL PoPx groups for cell cycle testing were formed. The experiments were repeated 3 times for these in vitro analysis.
Annexin V assay
Muse Cell Analyzer device and compatible Muse Annexin V kit and dead cell assay reagent were used for apoptosis analysis (Millipore; MCH100115).
MDA-MB-231 cells were cultivated in 24-well plates with 1 × 105 cells per well and left to incubate for 24 and 48 h. Then, cells were treated with trypsin and stained with annexin V and dead cell reagent according to the manufacturer’s protocols. (Millipore Corporation) and analyzed using Muse Cell Analyzer (Millipore Corporation).
Cell cycle assay
MuseR Cell Cycle Kit were used for detect the cell cycle stage of cells (Millipore; MCH100106). MDA-MB-231 cells were cultivated in 24-well plates with 1 × 106 cells per well and left to incubate for 24 and 48 h. Later, cells were treated by removing with trypsin. After, the cells were stained with MuseR Cell Cycle Kit according to the manufacturer’s protocols (Millipore Corporation) and analyzed using Muse Cell Analyzer (Millipore Corporation).
AgNOR staining
Cultured MDA-MB-231 cells with 25 μg/mL and 50 μg/mL PoPx, control and treated group were spread on the clean slide and dried at room temperature. Later air dried, the slides were fixed in fixative (3 volume methyl alcohol:1 volume acetic acid) and the AgNOR staining method was carried out according to the slight modification of the protocol followed by Benn and Perle13 and Lindler.14 The AgNOR stained slides were seen with a light microscope (Leica DM 3000) and photographed with a digital camera (Imaging Color 12 BIT, Made in Canada). The captured images of MDA-MB-231 cell were transferred to image processing software (ImageJ version 1.47t, National Institutes of Health, Bethesda, Maryland, USA) and both total AgNOR area per nuclear area (TAA/NA) and mean AgNOR number was calculated via “freehand selection” tool for each nucleus. Fifty nuclei were determined for each slide. Each slides were counted by three other researchers and the average was taken. A demonstrative example of AgNOR staining of MDA-MB-231 cells was given in Fig. 1.
Fig. 1.
A demonstrative example of AgNOR staining of MDA-MB-231 cells at the end of 24 h (A: Control, B: 25 μg/mL PoPx, C: 50 μg/mL PoPx. ×20 magnification, Scale bar 200 mm, arrows shows argyrophilic nucleolar protein). AgNOR; argyrophilic nucleolar organizing region-associated protein.
Statistical analyses
The conformity of the data in the study to the normal distribution was evaluated with Shapiro–Wilk, histogram, and q-q graphs. One-way analysis of variance (ANOVA) was used for statistical comparison of parameters between groups. The homogeneity of the variances of the data was evaluated using the Levene test. Tukey test was used for multiple comparisons. For each group, the differences between the 24-h and 48-h measurements were evaluated with the paired t-test. The analysis of the data was carried out in Turcosa (Turcosa Analytics Ltd Co, Turkey, www.turcosa.com.tr) statistical software. p-values less than 0.05 were deemed significant in statistics.
Results
Total phenolic substance and punicalagin amount
As a result of the analysis made by HPLC method, 1,235 μg phenolic component was found in 50 g (250 mL) PoPx. It was determined that the most abundant phenolic substance in the pomegranate peel was punicalagin with 70%. Table 1 shows the total phenolic substance and punicalagin content of the extracts used in our study.
Table 1.
Amount of total phenolic substance and punicalagin contained in extract amounts used in the study.
| Amount of pomegranate peel extract | Total amount of phenolic substances | Amount of punicalagin |
|---|---|---|
| 25 μg | 0.6175 μg | 0.4323 μg |
| 50 μg | 1,235 μg | 0.8645 μg |
Annexin V and dead cell test results
In the graphics created by the Muse device, four partitions formed by the intersection of the horizontal and vertical axes were observed. Cell populations formed by the markers in the test were observed in these compartments. Dead cells were collected in the left upper quadrant, living cells in the left lower quadrant, late apoptotic cells in the right upper quadrant, and early apoptotic cells in the right lower quadrant. Data graphs obtained from Muse Cell Analyzer device at the end of 24 and 48 h incubation periods are shown in Fig. 2. In the control groups were evaluated in terms of the number of viable cells depending on the incubation times, there was a statistically significant difference between the 24-h and 48-h incubation times p < 0.05), while there was a statistically significant difference between the 24-h and 48-h incubation times of the other groups. no difference was found (p > 0.05).
Fig. 2.
1-Data graphics obtained from muse cell analyzer device, showing apoptosis rates of MDA-MB-231 cells by groups at the end of the 24 h ıncubation period A. control group, B. 25 μg/mL pomegranate group, C. 50 μg/mL pomegranate group. 2-Data graphs obtained from muse cell analyzer, showing apoptosis rates of MDA-MB-231 cells by groups at the end of the 48-h ıncubation period a. control group, B. 25 μg/mL pomegranate group, C. 50 μg/mL pomegranate group.
When the results were evaluated depending on the dose at the end of the 24-h incubation period, it was determined that there was a significant decrease in the number of viable cells in both 25 μg/mL PoPx and 50 μg/mL PoPx groups compared to the control (p < 0.001). When the results were evaluated depending on the dose at the end of the 48-h incubation period, it was determined that there was a statistically significant difference in the decrease in the number of viable cells in both the 25 μg/mL PoPx and 50 μg/mL PoPx groups compared to the control group (p = 0.001).
When the results were evaluated depending on both the duration and the dose, it was observed that the percentage of viable cells of the 50 μg PoPx group with a 48-h incubation period was significantly lower than the percentage of viable cells of the other groups (p < 0.05).
When the percentage of early apoptotic cells were evaluated, depending on their incubation times, it was noted that there was a statistically significant difference between the 24-h and 48-h incubation times of each group (p < 0.05). When the results were evaluated depending on the dose at the end of the 24-h incubation period, an increased early apoptosis value was observed in both 25 μg/mL PoPx and 50 μg/mL PoPx groups compared to the control (p < 0.001). It was observed that there was a significant increase in all groups (p < 0.001). When the results were evaluated depending on both time and dose, it was observed that the percentage of early apoptotic cells in the 50 μg PoPx group with 48 h incubation period was significantly higher than the percentage of early apoptotic cells in the other groups (p < 0.05).
When the percentage of late apoptotic cells were evaluated, depending on their incubation times, it was noted that there was a statistically significant difference between the 24-h and 48-h incubation times of each group (p < 0.05). When the results were evaluated depending on the dose at the end of the 24-h incubation period, an increased late apoptosis value was observed in both 25 μg/mL PoPx and 50 μg/mL PoPx groups compared to the control group (p < 0.05), whereas late apoptosis was observed in 48 h results. It was observed that there was a significant increase in the values of all groups compared to the control group (p < 0.05).
When the groups were evaluated in terms of the percentage of total apoptotic cells, depending on their incubation times, it was noted that there was a significant difference between the 24-h and 48-h incubation times of each group (p < 0.05). The results were evaluated depending on the dose at the end of the 24-h incubation period, an increased total apoptosis value was observed in both 25 μg/mL PoPx and 50 μg/mL PoPx groups compared to the control group (p < 0.001). The total apoptosis values of the 48-h results were compared to the control group a significant increase was observed in all groups (p < 0.001).
Cell cycle test results
The data graphs obtained from the Muse Cell Analyzer device at the end of the 24 and 48 h incubation periods are shown in Fig. 2.
When the groups were evaluated depending on the incubation times, statistically significant differences were found between the 24 and 48 h culture results of the G0/G1 stage cell ratios of each group (p < 0.05). When the G0/G1 stage 24-h incubation results were evaluated, the rate of cells in the G0/G1 stage of the 25 μg/mL PoPx group was found to be significantly higher than the G0/G1 stage cell rates of the 50 μg/mL PoPx group and the control group (p < 0.05).
When the groups were evaluated according to the paired t test depending on the incubation times, statistically significant differences were found between the 24 and 48 h culture results of the S stage cell ratios of each group (p < 0.05). When the groups were evaluated according to the dose, it was found that there was no statistically significant difference between the S-stage cell ratios and the other groups compared to the control group (Fig. 3).
Fig. 3.
1-Muse analyzer device cell cycle test raw data charts by groups at the end of 24-h culture A. control group, B. 25 μg/mL pomegranate group, C. 50 μg/ml pomegranate group. 2- Muse analyzer device cell cycle test raw data charts by groups after 48-h of culture A. control group, B. 25 μg/mL pomegranate group, C. 50 μg/mL pomegranate group.
When the groups were evaluated depending on the incubation times, statistically significant differences were found between the 24-h and 48-h culture results of the G2/M stage data of each group (p < 0.05). While there was no statistically significant difference in the 24-h culture results evaluation of th G2/M stage data of the control, 25 μg/mL PoPx, 50 μg/mL PoPx groups, there was no significant difference between the groups in the 48-h culture results evaluation (p = 0.012).
Measurements were made from the photographs taken as a result of AgNOR staining. As a result of the analysis, AgNOR number (Table 2) and TAA/NA ratio (Table 3) at 50 μg/mL dose at the end of 24 h decreased compared to the control and were found to be statistically significant (p < 0.05). After 48 h, AgNOR number (Table 2) and TAA/NA ratio (Table 3) were not statistically significant between the groups (Fig. 4).
Table 2.
Mean AgNOR number after 24 and 48 h of incubation.
| Hours/Groups | Control | 25 μg/mL PoPx | 50 μg/mL PoPx | p |
|---|---|---|---|---|
| 24 h | 3,08 ± 0,89a | 2,66±1,02a,b | 2,56±1,01b | <0.001 |
| 48 h | 3,36±1,21 | 3,26±1,19 | 3,08±0,92 | >0.001 |
p < 0.05 was considered statistically significant. Data are expressed as mean ± SD (Standard deviation). There is no statistically significant difference between the groups containing the same letter (p > 0.05). AgNOR: Argyrophilic nucleolar organizer region. PoPx: Pomegranate peel extract
Table 3.
TAA/NA value at the end of 24 and 48 h of incubation.
| Hours/Groups | Control | 25 μg/mL PoPx | 50 μg/mL PoPx | p |
|---|---|---|---|---|
| 24 h | 0,06±0,03a | 0,06±0,32a,b | 0,04±0,02b | <0.001 |
| 48 h | 0,08±0,13 | 0,06±0,04 | 0,06±0,04 | >0.001 |
p < 0.05 was considered statistically significant. Data are expressed as mean ± SD (Standard deviation). There is no statistically significant difference between the groups containing the same letter (p > 0.05). There is statistically significant difference between the groups containing the different letter (p < 0.05). TAA/NA: Total AgNOR area (TAA)/Total nuclear area (NA) ratio. PoPx: Pomegranate peel extract.
Fig. 4.
A.-Comparison of AgNOR number and TAA/NA ratio between groups after 24 h of incubation. B. Comparison of AgNOR number and TAA/NA ratio between groups after 48 h of incubation.
Discussion
Cancer is a growing condition worldwide. Medicinal plants constitute a common alternative for cancer treatment. Antioxidative reagents have been applied in the treatment of many types of cancer. Pomegranate (PoPx) is a natural plant derived antioxidant and is a well-known fruit in this context, but its apoptotic effects property has not been extensively studied using AgNOR staining method and selection of the most accurate dose for cancer treatment. In the literature review, no researches about the association between AgNOR proteins amount and the effects of PoPx treatment have been performed on MDA-MB-231. Therefore, we showed the present study to indicate any possible effects of pomegranate (P. granatum L.) treatment on the NOR protein synthesis.
Bagheri et al.15 MDA-MB-231 cells were treated with different concentrations of PoPx for 24, 48 and 72 h.
In this study, the effects of PoPx on cell migration and invasion were determined by transwell assays and wound healing. To address the possible molecular mechanisms underlying the antimetastatic effect of PoPx, real-time quantitative PCR analysis of selected epithelial mesenchymal transition (EMT) markers were performed. Moreover, the expression of Î2-catenin as a critical factor in promoting cancer metastasis was examined. PoPx markedly inhibited the migration and invasion of cells at concentrations of 25, 50, 100, 250, 500, and 1,000 μg/mL. At relatively high concentrations (500, 1,000 μg/mL), PoPx induced apoptosis. Moreover, PoPx decreased the gene expression of vimentin, ZEB1, and Î2-catenin and also increased the expression of E-cadherin in TNBC cells. The protein level of Î2-catenin, as measured using western analysis, revealed a time-dependent decrease at the concentration of 1,000 μg/mL PoPx. Downregulation of EMT markers and Î2-catenin showed accordance with the inhibition of migration and invasion. The present data show that PoPx could be a promising drug candidate to reduce metastasis in TNBC cells. Especially pomegranate is a well-known fruit in this context, but its apoptotic effects using AgNOR staining property has not been extensively studied. Also in our study showed that the MDA-MB-231 cells were treated with 50 μg/mL PoPx group at 48th hour. Therefore, we showed from a different perspective that PoPx is effective at lower doses, in a shorter time and cheaper, with the AgNOR staining method.
Ahmadiankia et al.16 were investigated the effectiveness of PoPx on MDA-MB-231 breast cancer cell line, it was reported that exposure to PoPx killed breast cancer cells. It was found that PoPx significantly inhibited resistant cell migration, a protein essential for cell adhesion, up-regulated the expression of ICAM-1 and down-regulated the expression of MMP9, fibronectin and VEGF, which contribute to cancer cell migration.
Modaeinama et al.17 were tested PoPx in 4 different cancer cell lines including [A549 (non-lung non-small cell cancer), SKOV3 (ovarian cancer), MCF-7 (breast adenocarcinoma) and PC-3 (prostate adenocarcinoma)], 100, 250, 500, 1,000 μg/mL doses. It has been found that PoPx reduces cell viability below 40% in all cancer cells studied, even at the lowest doses.
Keta et al.18 were investigated the effect of PoPx on different human cancer cell lines (MCF7, HTB140, HCT116, HTB177) and MRC-5 normal fibroblasts; showed that PoPx expressed selective cytotoxicity for cancer cells compared to the normal cell line and exhibited good inhibitory effects on the cancer cell lines used.
Orgil et al.19 were examined the anti-proliferative activity of PoPx against breast (MDA-MB-453 and MCF7) and prostate (PC-3 and LNCaP) cancer cell lines, PoPx had high anti-inflammatory effects against MDA-MB-453, MCF7 and LNCaP cell lines. While its proliferative effect was observed, it was reported that it showed relatively high resistance in the PC-3 cell line.
Li et al.20 were showed the anticancer effect of PoPx on TPC-1 and BCPAP thyroid cancer cell lines, it was reported that the extracts strongly suppressed proliferation and induced cancer cell apoptosis in two types of thyroid cancer cell lines.
Ma et al.21 were performed that in a study conducted by PoPx polyphenols to observe the in vivo apoptotic and antiproliferative effects of pomegranate peel polyphenols on human prostate cancer cells (PC-3), it was determined that pomegranate peel reduced tumor volume and weight and significantly increased the rate of apoptosis in tumor-bearing nude mice.
Asmaa et al.22 were showed the effect of PoPx on myeloid leukemia cell line (K562), it was reported that in the cell cycle test performed after 72 h of culture, PoPx stopped the cell cycle in the G2/M phase and caused an inhibition in K562 cell proliferation.
Zhou et al.23 were reported that bladder cancer cell line, 100 μg/mL dose of PoPx was tried and cell proliferation was suppressed after 48 h of incubation.
Song et al.24 were reported that the effect of PoPx on HepG2 cells (a type of human hepatoma cell), it was concluded that pomegranate peel polyphenols can inhibit the growth of HepG2 cells by blocking the cell cycle and inducing the mitochondrial apoptotic pathway in a dose-dependent manner.
From past to present, phytochemical agents obtained from medicinal plants are used in the treatment of human disorders. Identification of new phytochemical agents and determination of the most reliable dose for cancer therapy are crucial to improve diagnostic accuracy and management of diseases. NORs are related to a majority of regulatory proteins and they have roles as functional subunits of the nucleolus.25 Alterations of AgNOR protein amounts reflect the metabolic activities and protein synthesis capacity of the cells. Different studies are performed on malign and benign lesions.26–28 In addition, there are several studies about the protective effects of phytochemical agents on cancer treatment and detection of the most reliable dose of these agents with AgNOR staining methods.10,11 Although the significant advances in the treatment and management of the disease, cancer-related deaths are still on the rise. Therefore, different treatment strategies such as phytotherapy using natural therapeutic agents such as pomegranate (P. granatum) may be used for disease treatment.
In our study, the effect of PoPx on cell viability and apoptosis in human MDA-MB-231 breast cancer cells depending on dose (25 and 50 μg/mL) and time (24 and 48 h) was investigated. When the relationship between PoPx and apoptosis of MDA-MB-231 cells was evaluated in the data obtained from Annexin V and dead cell test, it was found that PoPx had an inhibitory effect on cell proliferation by increasing apoptosis on MDA-MB-231 cells depending on time and dose. It was observed that the highest decrease in viable cell rate and the greatest increase in apoptotic effect were in the 50 μg/mL PoPx group at 48th hour. In the data obtained from the cell cycle test, a statistically significant difference was found between the 24-h results of the groups in the G0/G1 stage and the 48-h results of the groups in the G2/M stage (p < 0.05). In present study, we detected that mean AgNOR number and TAA/NA ratio of positive control is significantly higher than the MDA-MB-231 cell line treated with pomegranate (P. granatum L.).
Conclusion
In conclusion, the apoptotic effect of PoPx on MDA-MB-231 breast cancer cells was performed. According to the results we obtained; It was found that PoPx increased apoptosis on cancer cells by various mechanisms, inhibited cell viability/cell growth, and these effects were most evident in the 50 μg/mL PoPx group. In present study, we detected the apoptotic effect of pomegranate (P. granatum) on MDA-MB 231 derivate from human breast cancer. To obtain more accurate knowledge about the current research, additional studies including those on different metabolic pathways that have therapeutic features should be performed in various types of cancer. In this manner, good therapeutic approaches may be developed for making the management of diseases more accurate. Additionally, because this technique is simple, cheap, and serves as a valuable marker to evaluate the ribosomal gene activity in different metabolic durations of various cells, it has important advantages. It was detected that PoPx has an important role against cancer formation. The current study indicated that the detection of both the AgNOR values may be used also as a biomarker for detecting the success rate of the performed therapeutic strategy and selection of a reliable dose for accurate management of the disease.
According these results, we think that the widespread consumption of pomegranate (P. granatum) fruit peel may play an active role in reducing the risk of developing cancer, which is one of the biggest health problems of our time, in slowing the progression of the disease by using it as a complementary treatment in cancer treatment, and in improving the quality of life of patients by reducing the side effects caused by traditional treatment methods. There is a need for additional studies to support our results and explain the effects of PoPx totally and/or individually on cancer.
Contributor Information
Rabia Nur Ceyhan, Department of Nutrition and Dietetics, Faculty of Health Sciences, University of Nuh Naci Yazgan, Ertuğrul Gazi district, Nuh Naci Yazgan Campus, Küme Evler Kocasinan, Kayseri 38090, Turkey.
Mustafa Nisari, Department of Nutrition and Dietetics, Faculty of Health Sciences, University of Nuh Naci Yazgan, Ertuğrul Gazi district, Nuh Naci Yazgan Campus, Küme Evler Kocasinan, Kayseri 38090, Turkey.
Mehtap Nisari, Department of Anatomy, Erciyes University, Faculty of Medicine, Köşk district, Dede Efendi street, Melikgazi, Kayseri P.C:38030, Turkey.
Sümeyye Uçar, Department of Anatomy, Erciyes University, Faculty of Medicine, Köşk district, Dede Efendi street, Melikgazi, Kayseri P.C:38030, Turkey.
Fatih Mehmet Koca, Department of Anatomy, Erciyes University, Faculty of Medicine, Köşk district, Dede Efendi street, Melikgazi, Kayseri P.C:38030, Turkey.
Gülderen Kerek, Department of Anatomy, İstinye University Faculty of Medicine, Hamidiye, Kağıthane İstanbul 34408, Turkey.
Tuğçe Özcanlı, Department of Anatomy, İstinye University Faculty of Medicine, Hamidiye, Kağıthane İstanbul 34408, Turkey.
Neriman İnanç, Department of Nutrition and Dietetics, Faculty of Health Sciences, University of Nuh Naci Yazgan, Ertuğrul Gazi district, Nuh Naci Yazgan Campus, Küme Evler Kocasinan, Kayseri 38090, Turkey.
Author contributions
Study conception and design: Mustafa Nisari; data collection: Rabia Nur Ceyhan, Mehtap Nisari, Sümeyye Uçar, Tuğçe Özcanlı, Gülderen Kerek, Fatih Mehmet Koca; analysis and interpretation of results: Mustafa Nisari, Rabia Nur Ceyhan, Mehtap Nisari, Neriman İnanç; draft manuscript preparation: Mustafa Nisari and Rabia Nur Ceyhan. All authors reviewed the results and approved the final version of the manuscript.
Funding
This study was supported by Nuh Naci Yazgan University Scientific Research Projects Unit (BAP) (Project code: 2021/SA-LTP-2).
Conflict of interest statement. The authors declare that they have no conflicts of interest.
Ethics approval and consent to participate
Ethics committee approval is not required as the study did not include human and/or animal studies.
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