|Year : 2019 | Volume
| Issue : 3 | Page : 195-204
Cytotoxic effects of juglone and Pterocarya fraxinifolia on prostate cancer cells
Morteza Mahdavi1, Mohammad Azadbakht2, Akbar Vahdati1, Mohammad Shokrzadeh3, Ayub Farhadi4
1 Department of Biology, Shiraz Branch, Islamic Azad University, Shiraz, Iran
2 Department of Pharmacognosy and Biotechnology, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
3 Department of Toxicology and Pharmacology, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
4 Department of Animal Science, Laboratory of Molecular Genetics and Animal Biotechnology, Faculty of Animal Sciences and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran
|Date of Web Publication||9-Jul-2019|
Dr. Mohammad Azadbakht
Department of Pharmacognosy and Biotechnology, Mazandaran University of Medical Sciences, Sari
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Juglone with naphthoquinone structure has medicinal properties and its anticarcinogenic and antioxidant effects have been proven. In this research, the cytotoxic and apoptosis effects of juglone and Pterocarya fraxinifolia (PF) methanolic extract on human prostate cancer cells were studied. Materials and Methods: The PC3 and DU145 human cancer cells and normal cells of primary prostate epithelial cells (ATCC PCS-440-010) were treated with juglone and PF extract at the concentrations of 10, 50, 100, 500, and 1000 μg/mL for 24, 48, 72, and 96h. The morphological changes were examined by reversed microscope. The survival percentage of cell lines was evaluated by MTT (3,4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide) test. The rate of apoptosis and expression of AR and CLU genes were examined by flow cytometry and real-time polymerase chain reaction. Results: All concentrations after 24h caused morphological changes in PC3 and DU145 cells, and these changes were intensified after 48, 72, and 96h. Also, concentrations of 100 and 500 μg/mL caused morphological changes in normal cells. The results of MTT test showed a significant decrease in PC3 and DU145 cell survival rate at 50, 100, and 500 μg/mL concentrations (P < 0.05). Juglone at 10 μg/mL concentration induced apoptosis in cancer cell lines. Conclusion: Juglone and PF could decrease the growth of cancer cell lines through the mitochondrial pathway. So PF could be considered as a potential candidate for therapeutic herbal medicine in treating cancers.
Keywords: Apoptosis, cytotoxicity, juglone, methanolic compound, prostate cancer cells, Pterocarya fraxinifolia
|How to cite this article:|
Mahdavi M, Azadbakht M, Vahdati A, Shokrzadeh M, Farhadi A. Cytotoxic effects of juglone and Pterocarya fraxinifolia on prostate cancer cells. J Pharm Bioall Sci 2019;11:195-204
|How to cite this URL:|
Mahdavi M, Azadbakht M, Vahdati A, Shokrzadeh M, Farhadi A. Cytotoxic effects of juglone and Pterocarya fraxinifolia on prostate cancer cells. J Pharm Bioall Sci [serial online] 2019 [cited 2020 Apr 3];11:195-204. Available from: http://www.jpbsonline.org/text.asp?2019/11/3/195/262191
| Introduction|| |
Androgen receptor (AR) transporter plays a pivotal role in the development, physiology, and pathology of prostate gland. After attaching its internal ligands, which include 5α-dihydrotestosterone and testosterone, the main function of the AR is as a transcription regulator in regulating gene expression., In the nucleus, the activating ligands of ARs are known as DNA-sequencing assistant, such as the androgen response elements, a nuclear regulatory partner, and transcriptional machine, and thus play a role in regulating the transcription of target genes.,, AR carriers are a center for regulating prostate growth, cell differentiation, and homeostasis, and the false regulation of AR carriers leads to the onset and progression of prostate cancer. More studies of prostate cancer cells show the effect of AR on growth and development of these cells., It is believed that the increased expression of AR in luminal epithelial cells causes suppression of proliferation and preservation of its secretion function. In addition, the mediastinal AR mediator in mesenchymal/stroma and the paracrine carrier mechanism can also regulate the survival of luminal epithelial cells in the prostate. Although the proliferation of AR-regulated cells has been widely studied, little has been known about the response of cellular stress and apoptosis of prostatic epithelial cells to the role of AR carriers, which play a central role in prostate gland growth and homeostasis. Apoptosis is highly regulated, a process in which carriers are characterized by cellular death in an energy-dependent manner., The imbalance between proliferation and cell apoptosis leads to cancer. Bcl-2 genes are regulating apoptosis and Bax overexpression as proapoptotic protein induces apoptosis. The Bcl-2 protein family are involved in prostate cancer; also, the AR carrier plays an important role in the prostate epithelial homeostasis, which depends on the net cell division. However, AR carriers and the stress response of apoptosis in human prostate epithelial cells are not well known.Pterocarya fraxinifolia (PF) is a tree species that belongs to Juglandaceae family, also known as “Caucasian wingnut” or “Caucasian walnut” in English and “Larg” in Farsi [Figure 1].
|Figure 1: Pterocarya fraxinifolia tree at Hizarjarib forest, Neka city, north of Iran|
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PF is native to Armenia, Azerbaijan, Georgia, Iran, Russia, Ukraine, and Turkey. Its leaves are used ethnobotanically as fish poison, for dyeing, and as antidiarrheal agent. The plant consists of phenols, flavonoids, and naphthoquinone especially 5-hydroxynaphthoquinone (juglone), which represent antimicrobial, antioxidant, and cytotoxic activities. In this research, the apoptotic effect of PF and juglone on PC3 and DU145 prostate cancer cell lines was examined by MTT (3, 4, 5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide) assay and flow cytometry. Also, the expression of mRNA and hypermethylation of DNA were determined by real-time polymerase chain reaction (PCR).
| Materials and Methods|| |
Plant collection and extraction
The PF leaves were collected from Hizarjarib forest in Neka city at Mazandaran province, north of Iran. The plant was systematically indentified by Prof. Mohammad Azadbakht (Department of Pharmacognosy, Mazandaran University of Medical Sciences, Sari, Iran). The leaves were dried under standard conditions at 40°C away from light. The dried leaves were powdered and macerated with methanol solvent. The extract was concentrated by rotary evaporator.
The PF extract was assayed based on juglone using high-performance liquid chromatography (HPLC) apparatus (Knauer-YF 50-C18, 100A column [250 mm× 4.6mm with C18 precolumn] plus quaternary gradient pump (PU-2089) plus UV-E4310 intelligent UV/vis detector 255nm) and at a flow rate of 1mL/min with 10-min retention time. The methods of isocratic mobile-phase conditions were as follows: 50% acetonitrile: 50% 0.01 M sodium dihydrogen phosphate, pH 5.5.
PC3 and DU145 cell lines and normal primary prostate epithelial cells were cultivated in RPMI containing l-glutamine, penicillin (100 μg/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum. Cells were incubated at 37°C, 90% moisture content, and 5% carbon dioxide. In a morphological study, concentrations of 10, 50, 100, 500, and 1000 μg/mL of juglone methanolic compound were used on cultured cells (105 × 5) in sterile filtered 50-mL lid flasks (JET BIOFIL, Canada), which contained 5 mL of the culture medium at 24, 48, 72, and 96 h after treatment of the cells and were photographed using an inverted microscope equipped with a camera (OLYMPUS, Japan) at each of the designated hours. MTT colorimetric method was used to evaluate the toxicity of methanolic compound of zygon on both PC3 and DU145 cell lines and normal primary prostate epithelial cells. This method is a mitochondrial metabolic test and is based on the breakdown of tetrazolium salt by the mitochondrial succinate dehydrogenase enzyme of living cells. After each passage, a cell suspension was prepared in a culture medium. In this way, 96 houses of 5000 cells in each house are available. For this test, 3 plates of 96 houses were considered and 30 houses of each plate were selected. Then, five groups of dwellings were assigned to PC3 and DU145 cells and three groups of trichomes from the house to the primary cells of the primary prostatic epithelial cells. Plates were placed in an incubator for 24h to adhere to the cells. Then the culture media of all three empty plates and 180 μL of fresh culture were laid in each house. Concentrations of 10, 50, 100, 500, and 1000 µg/mL of jelly on sterilized compound were added to 4 groups of 3 doses of PC3 and DU145 cells and natural primary prostate epithelial cells, and were transferred to home. The control group was added to the size of the composition, Gibco sterile distilled water. Therefore, three plates with these specifications were obtained. Five groups of three houses from each plate containing 10, 10, 50, 100, 500, and 1000 μg/mL concentrations of PC3 and DU145 cells and five groups for three cells containing these concentrations of normal cells of primary prostate epithelial cells were considered. The plates were placed in an incubator. After 24 h, one of the dishes was removed, and the culture medium was discarded. The plate was then added 180-μL fresh medium with 20-μL MTT and was kept for 4h in an incubator. Subsequently, the culture medium containing the empty MTT color, 200-μL DMSO, and 25-μL glycine buffer was replaced and the light absorption of the house was measured by the Elisa reader at StatFax 303 Plus at a wavelength of 570nm. The latter steps were performed for the second plate after 48h and for the third plate after 72h after treatment of the cells with methanolic juglone combination.
The rate of DNA cleavage as a biomarker of apoptosis was determined by agarose gel electrophoresis method. PC3 and DU145 were cultured in small culture flasks for 24h. Ward cell numbers in suspension containing 5 × 105 cells, 3 mL of was transferred to 6-well cell culture plates. The cells were treated with PF extract and juglone separately and incubated with CO2 for 48h. Non-treated cells were considered as control group. The cells were collected and washed with phosphate-buffered saline (PBS). The DNA was purified using DNA purification kit, electrophored on 1.5% agarose gel, stained by ethidium bromide dye and visualized under gel documentation device (BioRad).
The quantitative assessment of apoptosis was determined by Annexin V-FITC kit.
Suspension was treated with PF extract, juglone, and cisplatin as positive control separately and was incubated for 24, 48, and 72h. The cells were washed with cold PBS twice and suspended in binding buffer. Five milliliters of FITC-conjugated Annexin V and 5mL of propidium iodide were added to 100mL of the cells and incubated for 15min at 25°C at dark. The colored cells were diluted by the binding buffer and were analyzed using flow cytometer.
Ten milliliters of Triazol (Invitrogen, USA) was added to treated DU145 and PC3 flasks and incubated at 25°C for 5min. Chloroform (400 µL) was added and centrifuged (g ×12000) at 4°C. The aqueous layer was mixed with 500 µL isopropanol and recentrifuged. Ethanol (70°C) was added to precipitated RNA and centrifuged (g ×7500) at 4°C. The extracted RNA was dissolved in diethylpyrocarbonate water.
Relative gene expression using real-time PCR
The AR gene on chromosome X and CLU gene on chromosome 8 were considered as goal and reference gene, respectively. Primers for amplification of AR gene were designed using Oligo 7 software according to NC_000023.11 accession number and their sequence homology was checked using BLAST (http://www.ncbi.nlm.nih.gov/blast). Primer sequences were: F-5/-GAAGTTGTATCTCTGTAGGTG and R-5/-AAGAGGAGTAACTAATGTGGAT (for AR); F-5/-GCTCTCCAGAACATCATCC and R-5/-GATGTCATCATATTTGGCAGG (for GAPDH). The total gene specificity of the nucleotide sequences was confirmed by BLASTN. Both the AR and GAPDH primers showed no significant sequence similarity to each other, or to other known human gene sequences, as assessed by a GenBank database search. The real-time PCR was performed using ABI 7300 (Applied Biosystems; Foster City, CA, USA) model and SDS (Sequence Detection System), ver 3.2.1, software. The amplification curve was plotted for each reaction.
DNA methylation detection
The DNA genome form cultured cells were extracted using Wizard Genomic DNA Purification kit and sodium bisulfide (Qiagen, Hilden, Germany) followed by the manufacturer’s protocols.
Data were analyzed by SPSS software (version 20) using one-way analysis of variance. The Tukey post hoc test was used at the significant level of P < 0.05.
| Results|| |
[Figure 2] shows the presence of Juglone in the extract of Pterocarya fraxinifolia with HPLC.
|Figure 2: The HPLC chromatogram of PF extract and juglone as reference standard|
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In the morphological evaluation of two PC3 and DU145 cell lines, there was no significant change in the 24h after the addition of methanolic juglone compound at 10 μg/mL concentration compared to control group cells [Figure 3]. However, in 10, 50, 100, 500, and 1000 μg/mL concentrations, the cells were isolated from their substrate and spiked in spindle form and cellular granulation was observed. The severity of the fecundity of the juglone methanolic composition was concentration dependent and increased with increase in fecundity. By increasing the concentration, the increase in the number of damaged cells was seen in the culture flasks (1,000 < 10,000). After 48, 72, and 96h of addition of methanolic compounds (50 μg/mL concentration), no significant change was observed in the flask cells in the control group, but at the concentrations of 10, 50, 100, 500, and 1000 μg/mL, changes in the morphology were observed. These changes became more intense with increasing time. At each concentration of 10, 50, 100, 500, and 1000 μg/mL, the effect on cells increased with increase in cytotoxic activity of the cells (cancerous and natural), and the number of cells killed was also found to be increased (48 < 72 < 96). In morphological evaluation of normal cells of l-primary prostatic epithelial cells, after 24h, reduction in division and cellular granulation was observed after addition of zygonone composition at 100, 500, 1000 μg/mL concentrations. However, at 10 and 50 μg/mL concentrations, cells were retained to the flask bed, retaining their normal shape and not showing cellular granulation. After 48, 72, and 96h, the effects of the compound increased at 100, 500, and 1000 μg/mL concentrations.
|Figure 3: PC3 and DU145 cancer cells, 24, 48, 72 and 96h after treatment with methanolic combination of juglone (×20). (A) Control; (B) 10 μg/mL after 24h; (C) 10 μg/mL after 48h; (D) 10 μg/mL after 72h; (E) 10 μg/mL after 96h; (F) 100 μg/mL after 24h; (G) 100 μg/mL after 48h; (H) 100 μg/mL after 72h; (I) 100 μg/mL after 96h; (J) 500 μg/mL after 24h; (K) 500 μg/mL after 48h; (L) 500 μg/mL after 72h; (M) 500 μg/mL after 96h; (N) 1000 μg/mL after 24h; (O) 1000 μg/mL after 48h; (P) 1000 μg/mL after 72h; and (Q) 1000 μg/mL after 96h. Arrows indicate the morphological changes in which the normal shape of cells (spindle shape) has become rounded and smaller. Also, cellular granulation was observed in them and this condition is a sign of the death of cells|
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The light absorption from the house containing cells treated with juglone methanolic combination was compared with the light absorption of the house containing the control group, and the percentage of surviving cells was calculated by the following formula:
The results showed that the PF extract at 100 and 500 μg/mL concentrations compared with the control group significantly reduced the survival rate of PC3 and DU145 cancer cells [Table 1]. At a concentration of 10 μg/mL of juglone combination, no significant decrease was observed in the survival rate of PC3 and DU145 cancer cells compared to the control group (P < 0.05). Compared to the control group, 500 and 1000 μg/mL concentrations of juglone combination significantly reduced the percentage of survival of normal cells of the primary prostate epithelial (P < 0.05), whereas at 10, 50, and 100 μg/mL concentrations of juglone combination, the reduction in the percentage of survival of normal cells of the primary prostate epithelial was not significant compared to the control group [Table 2].
|Table 1: Survival rate of DU145 cells at 24, 48, and 72 h after treatment with Pterocarya fraxinifolia methanolic extract at concentrations of 10, 50, 100, 500 and 1000 μg/mL|
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|Table 2: The effect of Pterocarya fraxinifolia methanolic extract on PC3 cells at different concentrations and exposure times|
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Rate of apoptosis
The apoptosis of DU145 and PC3 caused by juglone at a concentration of 100 µg/mL was 91.75% and 8.53%, respectively, in comparison to cisplatin, 12.18% and 19.82%, respectively [Figure 4] and [Figure 5]. In treated cells, significant increases (P < 0.05) marked by asterisks in percentage distribution of cells in early (0.32%) and late (91.43%) apoptosis were observed. In treated cells, significant increases (P < 0.05) marked by asterisks in percentage distribution of cells in early (0.60%) and late (7.93%) apoptosis were observed.
|Figure 4: (A) Apoptosis analysis of DU145 cells treated with IC50 concentration of juglone for 24h using annexin-V FITC and propidium iodide (PI) staining. (B) Flow cytometric scans of untreated cells showed only 4–8% of cells in early or late apoptosis stage|
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|Figure 5: (A) Apoptosis analysis of PC3 cells treated with IC50 concentration of juglone for 24h using annexin-V FITC and propidium iodide (PI) staining. (B) Flow cytometric scans of untreated cells showed only 9–10% of cells in early or late apoptosis stage|
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Expression of AR gene
The data of real-time PCR have shown that juglone at a concentration of 100 µg/mL increased the expression of AR gene in DU145 by 91.75% and by 64.51% in PC3, whereas cisplatin by 9.69% in PC3 and by 5.48% in DU145 [Figure 6].
|Figure 6: Expression of AR gene in DU145 and PC3 cells after treatment with juglone|
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| Discussion|| |
The main objective of this study was to investigate the cytotoxic effects of juglone methanolic combination on the PC3 and DU145 cancer cells in comparison with normal primary prostate epithelial cells. On the basis of the morphological observations, no significant morphological changes were observed 24h after treatment of cancer cells and normal cells with juglone methanolic combination at 50 μg/mL concentration compared to the control group, and the cancerous and normal cells were found to be similar in shape and size with control group cells. 48 and 72h after the cancerous and normal cells were affected by the extract at a concentration of 50 μg/mL, the morphological changes in them were very compared exactly with the control group. On the basis of the results of the MTT test, the reduction in the survival rate of PC3 and DU145 cancer cells and normal primary prostate epithelial cells 24h after treatment with a compound of 50 μg/mL concentration was not significantly different compared to the survival rate of samples in the control group (P < 0.05) [Table 3]. By increasing the time, the survival rate of cancerous and normal cells decreased, but this was not significant (P < 0.05). Therefore, increasing the time to 24h did not significantly reduce the survival rate of the cells exposed to the compound at a concentration of 10 μg/mL. According to the morphological observations, 24h after the treatment of PC3 and DU145 cancer cells with juglone compound at a concentration of 50 μg/mL, significant morphological changes were observed in the cells compared to the control group, and these changes intensified 48 and 72h after the treatment [Table 3] and [Table 4]. On the basis of the results of the MTT test, the survival rate of PC3 and DU145 cancer cells decreased significantly 24h after treatment with juglone compound at a concentration of 20 μg/mL compared to the control group (P < 0.05). Also, this decrease significantly increased with increase in time to 24h [Table 5] and [Table 6]. Therefore, it can be said that, in addition to the fact that the concentration of the juglone compound is effective on the morphology and survival rate of cancer cells, the duration of exposure to the cells can be effective. Compared to the control group, no significant morphological changes were observed 24h after the treatment of normal primary prostate epithelial cells with 50 μg/mL concentration juglone compound, and the MTT test confirmed these results. Reduction in the survival rate of normal primary prostate epithelial cells was not significant 24h after the treatment of the cells with the compound at a concentration of 100 μg/mL compared to the control group (P < 0.05). Also, increasing the time to 24h did not have significant effects on the morphology and survival rate of normal cells at 50 μg/mL concentrations. Therefore, the juglone compound at this concentration can significantly reduce the survival rate of cancer cells compared to the control group, but this decrease was not observed in normal cells. According to the research method used of this study, molecular differences between the cancerous and normal cells that lead to different responses of these two cell lines according to juglone compound at 100 μg/mL concentration cannot be investigated and detected. However, cancerous and normal cells differ in terms of gene expression pathways, external and intracellular receptors, as well as cellular signaling pathways, which may lead to different normal cell responses, compared to cancerous cells at a concentration of 100 μg/mL. This difference in signaling pathways in cancerous and normal cells may lead to the activation of apoptosis and inhibitors of DNA replication, or lead to the inhibition of a specific gene that has a regulatory role in the cell division cycle in cancer cells. The morphological changes at concentrations of 500 and 1000 μg/mL increased with increase in the concentration compared to the control group, and increase in the time to 24h after treatment with the compound could lead to intensification of cytotoxic effects in cancerous and normal cells. On the basis of the results of the MTT test, the survival rate of cancerous and normal cells at 500 and 1000 μg/mL concentrations significantly decreased (P < 0.05). Also, the survival rate of normal and cancerous cells at concentrations of 500 and 1000 μg/mL depends on the concentration and time. Increase in these factors increase the mortality rate of the cancerous and normal cells significantly. There is currently no report on the cytotoxic effects of juglone methanolic combination on PC3 and DU145 cancer cells and on the normal primary prostate epithelial cells. Therefore, the cytotoxic effects of the juglone compound on normal cells should also be considered and further research should be carried out in this regard. More consideration may be needed when using different concentrations of this herb, if the juglone compound has an inhibitory and cytotoxic effect on healthy human cells. Nakagawa et al. reported in 2001 that induction of programmed cell death is one of the most important mechanisms of the anticancer effects of the juglone compound.
|Table 3: The effect of Pterocarya fraxinifolia methanolic extract on normal prosthetic epithelial cells at different concentrations and exposure times|
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|Table 4: Survival rate of DU145 cells at 24, 48, and 72h after treatment with juglone methanolic combination at concentrations of 10, 50, 100, and 500 μg/mL|
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|Table 5: Survival rate of PC3 cells at 24, 48, and 72h after treatment with juglone methanolic combination at concentrations of 10, 50, 100, and 500 μg/mL|
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|Table 6: Survival rate of human primary prosthetic epithelial cells at 24, 48, and 72h after treatment with juglone methanolic combination at concentrations of 10, 50, 100, and 500 μg/mL|
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Zhang et al. found that juglone from Juglans regia methanolic extract stopped and inhibited the activities of three important metabolic processes, namely the production of RNA, DNA, and protein in human cancer cells. The inhibitory effect of juglone methanolic combination on nucleic acid synthesis could be a biochemical basis for its inhibitory effect on proliferation of tumor cells. This was investigated by testing the effects of juglone on the production of RNA and DNA in a cell-free system in which cores were isolated.
Wu et al. showed that juglone decreased the proliferation of tumor stem-like cells and induced apoptosis significantly with increasing caspase 9 cleavages. Also, juglone stimulated the survival of U87-bearing mice in comparison to control group.
In a study by Ji et al., the IC50 of juglone for gastric cancer SGC-7901 cells were 36.51±1.05 μmol/L and 25.37±1.19 μmol/L after 24 and 48h, respectively. Juglone induced apoptosis and the downregulation of Bcl-2 and upregulation of Bax via a mitochondrial pathway. Xu et al. demonstrated that the IC50 of juglone for HL-60 cells is approximately 8 μM. The probable cytotoxic effect of juglone is apoptosis induction in cancer cells through P53-dependent pathways and P53-independent pathways. In general, the methanolic extract of PF can reduce cell proliferation and division, and by activating the process of apoptosis in cells and also by damaging DNA and disrupting cellular signaling processes, which leads to destruction and death of the cell.
| Conclusion|| |
According to the results of this study, the juglone methanolic combination at a concentration of 50 μg/mL can kill cancer cells without causing significant damage to normal cells. It is hoped that this study will provide a framework for obtaining compounds from the juglone combination that can specifically target cancer cells.
Appreciation and gratitude to all the respected professors who helped me with this research, especially friends and colleagues at the Cellular Evolution Research Laboratory.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]