Abstract
Background/Aim: Drug resistance is a significant cause of high mortality in ovarian cancer (OC) patients. The reverse transcriptase inhibitor azidothymidine (AZT) has been utilized as a treatment for tumors, but its role in OC treatment has not been revealed. The aim of the present in vitro study was to examine the influence of AZT on the growth of human OC cells and the involved proteins. Materials and Methods: The proliferation, cell cycle distribution, extent of apoptosis, mitotic index, and terminal restriction fragment length were examined in three OC cell lines, CaOV3, TOV112D, and TOV21G, treated with AZT. Results: AZT inhibited growth of the TOV21G and CaOV3 cell lines by regulating cell cycle distribution. Specifically, AZT caused G2/M phase arrest on TOV21G cells and S phase arrest on CaOV3 cells. In addition, AZT treatment induced up-regulation of p21 and p16 in the TOV21G and CaOV3 cell line, respectively. Conclusion: AZT inhibited cell proliferation in serous and clear cell OC via the regulation of cell cycle distribution.
Ovarian cancer (OC) has the highest mortality among gynecological cancers and is the fifth leading cause of cancer-related death in women (1). The global incidence of OC was the seventh highest among gynecologic cancers in 2018 (2). Early diagnosis is difficult because OC is asymptomatic in its early stages, and nonspecific late-stage symptoms of this disease increase the difficulty of diagnosis. Consequently, OC is diagnosed at an advanced stage in more than 75% of affected women (3). Combination chemotherapy of paclitaxel and carboplatin is the conventional method to treat high-grade serous OC. Initially, the response rates are 60%-80%; however, eventually most patients become platinum-resistant, and subsequent relapses are observed (4). Although different combinations of drugs have been found to cure patients with platinum resistance, more than 70% of patients experience recurrence within 2 years (5). Identification of new drugs to prevent recurrence in chemotherapy-resistant patients is urgently required.
Zidovudine, also called azidothymidine (AZT), is a reverse-transcriptase inhibitor that was first used as an antiretroviral drug in the treatment of acquired immunodeficiency syndrome (6). However, AZT has also been reported to induce apoptosis or inhibit tumor cell growth in various human cancers, including breast (7), colon (8), lung (9), and ovarian (10) cancer.
Diverse biological mechanisms are involved in the AZT-mediated inhibition of cancer cell proliferation. AZT has been shown to affect the maintenance of telomere length in different types of carcinoma. Low dose of AZT has been reported to inhibit telomerase activity and enhance paclitaxel-induced apoptosis in hypopharyngeal squamous cell carcinoma (11). Moreover, AZT has shown to reduce the proliferation of colorectal cancer cells by inhibiting telomerase activity (8). Previous in vitro studies have shown that AZT induced cell cycle arrest in esophageal cancer, hepatoma, and mammary adenocarcinoma (12-14).
Until recently, only scant information concerning the use of AZT in the treatment of OC was available. Murakami et al. have demonstrated that AZT inhibited the growth of the human ovarian mucinous cystadenocarcinoma cell line, MCAS; this effect might depend on the regulation of p53 levels (15). In addition, AZT has been reported to induce cell death in OVCAR-3 cells from a high-grade human ovarian serous adenocarcinoma by up-regulating p53 and p14ARF expression (16). AZT has also been shown to inhibit cell growth by reducing the activity of telomerase in human ovarian serous cystadenocarcinoma SKOV-3 cells (17), as well as in the human ovarian serous cystadenocarcinoma cell line HO-8910 (18). Potentiation of AZT cytotoxicity was observed in cisplatin-resistant human ovarian carcinoma cells (10).
In this study, three types of human OC cells were treated with AZT and the effect on telomere maintenance, cell proliferation, cell cycle progression and effectors were investigated. The results suggested that AZT may possess the potential for clinical therapy for serous and clear cell OC.
Materials and Methods
Cell lines. Three human OC cell lines, CaOV3, TOV112D, and TOV21G, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (Gibco, Carlsbad, CA, USA), 1% L-glutamine, and 1% antibiotic solution of penicillin/streptomycin. AZT was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc (Kumamoto, Japan).
Cell proliferation assays. Cell proliferation rate and AZT dose–response studies were performed using the CCK-8 assay. The cells were seeded in 96-multiwell plates (3×103 cells/well) and treated with 0 to 2 mM AZT for the indicated durations. CCK-8 was added to the well for 4 h at 37°C. Then, absorbance was measured at 450 nm in a multiwell plate reader (TECAN, Mannedorf, Switzerland). The 20% and 50% inhibitory concentrations (IC20 and IC50) were determined using the forecast function of Excel.
Cell cycle and apoptosis assays. For examining cell cycle distribution, the cells were treated with indicated AZT doses for indicated durations. Live cells were collected, fixed with 70% cold ethanol, and treated with propidium iodine (Sigma–Aldrich) and PureLinkTM RNAse A solution (Invitrogen, Carlsbad, CA, USA). The stained cells were resolved on the FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). The distribution of each cell cycle phase was determined by plotting DNA content against cell number by using the FACSDiva™ software (Ver. 8.0.1). For apoptosis analysis, cells were harvested at indicated times and stained using reagents from the Annexin V-FITC apoptosis kit (BioVision, Milpitas, CA, USA). The stained cells were analyzed using the FACSCanto II flow cytometer (BD Biosciences) with CellQuest Pro software (Ver. 6.0).
Calculation of mitotic index. The cells were fixed and stained with the anti-phospho-histone H3 antibody. The cell nuclei were counterstained using 4’,6-diamidino-2-phenylindole (DAPI). The numbers of phosho-histone H3 positive cells and DAPI stained nuclei were estimated using ImageJ (Ver. 1.50i). The mitotic index was presented as the percentage of phospho-histone H3 positive cells divided by the total numbers of DAPI signal. At least 2,000 cells from 8 random fields were counted.
Terminal restriction fragment length measurement. Terminal restriction fragment (TRF) length measurement was performed using the TeloTAGGGTM Telomere Length Assay Kit (Roche, Mannheim, Germany). Eight micrograms of genomic DNA was digested with each 30 U Hinf I/Rsa I at 37°C for 2 h. The resulting fragments were fractionated by electrophoresis on 0.8% agarose gel and transferred to nylon membrane using Southern blotting technique. The transferred DNA was then fixed on the membrane by UV-crosslinking (120 mJ). The membrane was first pre-hybridized at 42°C for 1 h and then hybridized with a telomere-specific digoxigenin (DIG)-labeled probe at 42°C for 3 h. After washes with 2× saline-sodium citrate buffer, the membrane was incubated with anti-DIG-alkaline phosphatase. Finally, the immobilized telomere probe was visualized using alkaline phosphatase metabolizing CDP-Star (Roche), a highly sensitive chemiluminescence substrate. The membrane was then exposed to X-ray film, and the average TRF length was determined by comparing the signals with a molecular weight standard.
Western blotting. Whole cell lysates were extracted by RIPA buffer (APOLO, Qufu, PR China) according to its manufacturer's instructions. For measuring the indicated protein expression, 20 μg of total cell lysate was separated using a 4-20 gradient gel, and the proteins were detected using standard procedures. Western blots were incubated with primary antibodies against p21, CHK1, CHK2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ProteinTech, Chicago, IL, USA), AKT (Cell Signaling, Danvers, MA, USA), c-Myc, cyclin D1 (Novus Biologicals, Centennial, CO, USA), cyclin B1 (Invitrogen), cyclin A, p27 (Cell Signaling), p16, XPA, RPA1, ERCC1, GTF2H1 (GeneTex, Irvine, CA, USA), p53, and XPC (Santa Cruz). Blots were then incubated with corresponding horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoReasearch, West Grove, PA, USA) and detected using the ECL Western Blotting Substrate (Promega, Madison, WI, USA).
Transient transfection. The cells were seeded in a 6-cm plate one day before transfection, and a confluence of 60% was maintained. Next, 1 μg of control or gene-bearing plasmid DNA was incubated with Opti-MEM (Invitrogen) and mixed with 3 μl of ViaFect (Promega). Expression vectors pCMV3-N-OFPSpark-CDKN1A and pCMV3-CDKN2A, as well as the control vector pCMV3-N-OFPSpark, were purchased from Sino Biological (Chesterbrook, PA, USA). This mixture was incubated at room temperature for 15 min, and the cells were incubated with the mixture for indicated durations. p21-expression vector pCMV3-N-OFPSpark-CDKN1A and control vector were designed to be transfected into TOV-21G cells, while p16-expression pCMV3-CDKN2A and control vector pCMV3-N-OFPSpark were designed to be transfected into CaOV3 cells.
Statistical analysis. Our results are derived from at least three independent experiments and are reported as mean±standard error of the mean. Differences were analyzed using Student's t-test or one-way analysis of variance followed by the post-hoc Bonferroni or Dunnett test. All the statistical analyses were performed using SPSS, version 25. Differences with p-values of <0.05 were considered significant.
Results
AZT inhibited cell growth in human OC cells by regulating cell cycle distribution. To estimate the potential effects of AZT in the treatment of human OC, three types of OC cell lines CaOV3 (serous), TOV112D (endometriod), and TOV21G (clear cell) were included in this study. Initially, we calculated the IC20 and IC50 for each cell line by incubation with serially diluted concentrations of AZT. Our data showed that TOV112D was highly resistant (IC50=2 mM) to AZT; however, TOV21G (IC50=424 μM) and CaOV3 (IC50=25 μM; Table I) were relatively sensitive.
Determination of IC20 and IC50 values for AZT inhibition in human ovarian cancer cell lines.
We further analyzed the cell cycle distribution of three cell lines incubated with 0 μM (control), AZT IC20, and AZT IC50 for 72 h. In the TOV112D cells, the percentage of cells in the G1 phase decreased from 61.8 to 60.2 (p=0.95) and 58.1 (p=0.01) when treated with 0 μM, IC20, or IC50 AZT respectively (Figure 1A). In the TOV21G, the percentage of cells in the G2/M phase increased from 16.4 to 23.0 (p<0.001) and 33.4 (p<0.001) when treated with 0, IC20, or IC50 of AZT, respectively. This finding implies that AZT causes G2/M phase arrest in the TOV21G cell line (Figure 1A). The percentage of cells in the S phase increased from 19.1 to 36.3 (p<0.001) and 43.1 (p<0.001) in the CaOV3 cells when treated with 0 μM, IC20, or IC50 AZT, respectively. It is obvious that AZT caused cell cycle arrest at the S phase in the CaOV3 cell line (Figure 1A).
Since AZT inhibited cell growth in TOV21G and CaOV3 cell lines by arresting the cell cycle at specific phases, we further verified whether cells progressed to apoptosis after AZT treatment by using flow cytometry. Our results showed no apparent difference in apoptotic or necrotic ratio between the control and AZT-treated cells (Figure 1B).
AZT reduced the proliferation in TOV21G and CaOV3 cells. Since AZT affected the cell cycle distribution in the TOV21G and CaOV3 cells, we analyzed the cell mitotic index (MI) to confirm the inhibitory effect of AZT on cell proliferation. AZT significantly decreased the MI in the TOV21G cells from 2.38% to 1.26% (IC20, p<0.001) and 1.01% (IC50, p<0.001; Figure. 2A), as well as in the CaOV3 cells from 2.01% to 0.49% (IC20, p<0.001) and 0.14% (IC50, p<0.001; Figure 2A). No significant change of MI value was observed in the TOV112D cells (Figure 2A). These data implied that AZT inhibits cell proliferation at least in the two types of OC cells.
To verify whether AZT modulated cell cycle distribution by impairing telomere length (TL) maintenance, the changes in TL between AZT-treated cells and non-treated controls were estimated. Southern blot assay results showed no significant difference in TL between the control and AZT-treated cells in all three OC cell lines. The individual mean TL was 4.3, 32.1 and 2.26 kb for TOV21G, TOV112D, and CaOV3, respectively (Figure 2B).
AZT altered the expression of p21 and other proteins. To enhance our understanding of the proteins that were influenced by AZT treatment, we selected the moderately AZT-sensitive TOV21G cells for preliminary investigation. The cell cycle-related proteins that have been reported as the AZT downstream effectors, including cyclin D1 (12), p21 and p27 (19), CHK1, CHK2, cyclin A2 (20), cyclin A1 (21), and AKT1 and MYC (22), were analyzed through Western blotting. The expression levels of cyclin A2, cyclin D1, AKT, and myc were not affected by AZT treatment at any time point (Figure 3A). On the other hand, the expression levels of CHK1, CHK2, p16, p21 and 27 were influenced when cells were treated with AZT both in 48 and 72 h (Figure 3A). Furthermore, we examined the expression levels of proteins XPA, XPC, RPA1, GTF2H1, and ERCC1, which have been reported to be involved in the AZT-induced DNA damage (23). Western blot results showed no apparent variation in the expression levels of XPC, RPA1, GTF2H1, and ERCC1. XPA levels were slightly decreased by AZT treatment at 48 h, but not at 72 h (Figure 3B).
To further explore whether the same effectors in the AZT-treated TOV21G cells were also active in the CaOV3 and TOV112D cell lines, we examined the change in expression levels of CHK1, CHK2, p16, p21, p27, and p53 in the AZT-treated CaOV3 and TOV112D cells. Results showed that AZT effectively increased the expression of p16 in the CaOV3 cells (Figure 3C). However, the expression of p21 was apparently decreased in the AZT-treated cells (Figure 3C), and the levels of p27 were slightly decreased as well. The expression of CHK1 and CHK2, which was down-regulated in the TOV21G cells, remained constant. The expression of p53 protein was not detectable in the CaOV3 cells. In the AZT-resistant TOV112D cells, the expression levels of CHK1, CHK2, p27, p53, and p16 were not altered after exposure to AZT. Notably, the signal of p21 was extremely low in the TOV112D cells.
Statistical analysis showed that the expression of p21 was significantly up-regulated after AZT treatment of 48 h and maintained until 72 h (p<0.01) (Figure 3D, 3E). Down-regulation of CHK1 (p<0.01), CHK2 (p<0.01), p16 (p<0.01), and p27 (p<0.01) was also observed at 48 h (Figure 3D). Decrease of CHK1 (p<0.01), p16 (p<0.05), and p27 (p<0.01), but not CHK2 was observed at 72h (Figure 3E).
Azidothymidine (AZT) caused cell cycle arrest in a cell-type dependent manner. (A) Representative flow cytometry histograms show the cell cycle alterations at 72 h of AZT treatment with 0 μM, IC20, or IC50 in the three ovarian cancer (OC) cell lines. (B) Representative image of flow cytometry analysis of apoptotic and necrotic cell distribution in the three OC cell lines treated with 0 μM, IC20, or IC50 AZT for 48 h.
Azidothymidine (AZT) reduced mitosis in the TOV21G and CaOV3 cells but not in the TOV112G cells. (A) The cells were treated with 0 μM, IC20, or IC50 AZT for 48 h and fixed and stained with the anti-p-histone H3 antibody. The cell nuclei were counterstained with DAPI. The numbers of phospho-histone H3-positive and DAPI stained cells were estimated using ImageJ. The mitotic index is presented as the percentage of phospho-histone H3 positive cells divided by the total numbers of DAPI signal. At least 2000 cells from different fields were counted. (B) Representative image of telomere length, which was measured by Southern blotting of the terminal restriction fragments. Cells were treated with 0 μM, IC20, or IC50 AZT for 72 h.
Up-regulation of p21 protein caused G2/M phase arrest in TOV21G cells. The uncoordinated phenomena of p21 up-regulation and p16/p27 down-regulation in the AZT-treated TOV21G cells prompted us to further explore the role of p21 in TOV21G cells. For this reason, we transfected TOV21G cells to overexpress p21. Results showed that increased expression of p21 significantly reduced the cell distribution in the G1 phase from 69.7% to 62.7% (p<0.001). Simultaneously, the ratio G2/M significantly increased from 17.1% to 20.6% (p<0.05; Figure 4A). Western blot results indicated that the increase in p21 levels did not affect the expression level of p16 but apparently reduced the expression levels of p27 and slightly down-regulated the expression of CHK1 (Figure 4B).
AZT modulated the expression of cell cycle regulation–related genes in OC cell lines. (A) Western blotting analysis of cell proliferation and cell cycle regulation–related genes in TOV21G cells after 48 and 72 h of AZT treatment. (B) Western blotting analysis of DNA damage repair-related genes in TOV21G cells after 48 and 72 h of treatment with AZT. (C) Western blotting analysis of indicated genes in CaOV3 and TOV112D cells after 48 h of treatment with AZT. At least three independent experiments were performed; all showed the same patterns as represented figures. (D, E) Quantification of (A) CHK1, CHK2, p21, p16 and p27 levels normalized to GAPDH. Data are presented as mean±SD; Statistics were done by one-way ANOVA and Dunnett test. *p<0.05, **p<0.001 vs. control (Con) (D) and (E) represent for 48 h and 72 h respectively.
Up-regulation of p21 affected cell cycle distribution of TOV21G cells. (A) Representative images of flow cytometry analysis of cell cycle distribution in TOV21G cells transfected with pCMV3-N-OFPSpark control vector or pCMV3-OFPSpark-CDKN1A vector. Cells were fixed and analyzed at 48 h after transfection. (B) Western blotting analysis of indicated genes. Cells were treated as described in the text.
Up-regulation of p16 protein causes S phase arrest in CaOV3 cells. The same strategy was applied in the CaOV3 cells for the functional study of p16 levels. Up-regulation of p16 protein apparently reduced the distribution of cells in the G1 phase from 57.6% to 44.0% (p<0.001, Figure 5A). However, the percentage of cells in the S phase increased from 18.9% to 33.7% (p<0.001, Figure 5A). Western blotting results indicated that the protein expression of CHK1, p21, and p27 did not differ between the control cells and the p16-expressing CaOV3 cells (Figure 5B).
Discussion
Because of the high recurrence and chemotherapy-resistance of OC, an increasing number of studies have focused on the exploration of using new chemicals to overcome the occurrence of resistance to primary platinum-based chemotherapy. AZT, a reverse transcriptase inhibitor that has been used in the treatment of AIDS-associated Kaposi sarcoma, Epstein-Barr-associated lymphoma, primary central nervous system lymphoma, and adult T-cell leukemia (19), has been reported to reduce tumors in phase I and II clinical trials as an individual drug or in combination with other drugs for colorectal cancers, pancreatic cancer, and various advanced malignancies (24-27).
In the present study, three types of OC cell lines were exposed to AZT to evaluate the potential cytotoxic effect of this antiviral drug. We demonstrated that treatment with AZT significantly reduced OC cell proliferation in a dose-dependent manner in all cell lines tested, with diverse IC50 values. Cell cycle arrest and decreased MI data further confirmed the regulation of cell proliferation by AZT in the OC cells. However, apoptosis and TL shortening were not observed in the AZT-treated cells. These data indicated that AZT inhibited cell proliferation by regulating cell cycle; however, the detailed mechanism was not elucidated.
Up-regulation of p16 affects cell cycle distribution in of CaOV3 cells. (A) Representative images of flow cytometry analysis of cell cycle distribution in the CaOV3 cells transfected with pCMV3-N-OFPSpark control vector or pCMV3-CDKN2A vector. Cells were fixed and analyzed at 48 h after transfection. (B) Western blotting analysis of indicated genes. The cells were treated as described in the text.
Although AZT has considerable clinical benefits, serious side effects, particularly bone marrow suppression, limit the application of AZT in cancer therapy. Reduced drug dosage could effectively suppress toxic side effects and may enable the use of AZT in clinical cancer therapy. Our results showed that serous OC cells (CaOV3) were the most sensitive to AZT; the IC50 was approximately 25 μM. Previous evidence from the same type of serous OC cells, OVCAR-3, has shown that the effective concentration that causes cell death is approximately 1 mM for AZT (16). These diverse results imply that the presence of other factors, and not only tissue type, influence the effect of AZT on OC cells. AZT-induced cell apoptosis might occur via the p53-Puma/Noxa/Bax pathway and modulate cell cycles from the p53–p21 pathway (28). In our study, p53 protein could not be detected in the CaOV3 cells, and wild type p53 was not influenced by AZT in the TOV21G cells. We deduced that p53 is not involved in the AZT-induced OC cell death; however, the detailed mechanism has not be revealed.
In a previous study, it has been shown that AZT causes the S phase arrest in HeLa cells accompanied by the down-regulation of p21 (29). In accordance with this evidence, our findings showed that AZT induced S phase arrest and simultaneously reduced the expression of p21 in CaOV3 cells. On the other hand, up-regulation of p21 was observed when the G2/M arrest was induced by AZT in TOV21G cells. Similar to this finding, it has been previously reported that in AZT-induced cell cycle arrest in acute promyelocytic leukemia—induction of p21 expression attenuates arsenic trioxide–activated G2/M arrest (30). These inconsistent findings suggest that multiple gene regulation mechanisms, in which p21 is crucial, underlie AZT-induced cell cycle arrest.
The regulation of p21 expression can be modulated by a p53-dependent or independent pathway (31). According to the constant expression of wild-type p53 in TOV21G cells and the deficiency of p53 in CaOV3 cells, we deduced that the expression of p21 was modulated by a p53-independent pathway in AZT-induced cell cycle arrest. Results of a clinical study have shown that p21-positive staining was found more often on low-stage tumors, and p21-positive patients had a greater survival benefit than p21-negative patients (32). High expression levels of p21 have also been reported to be correlated with high survival rates in platinum-sensitive patients with OC (33). In this study, we demonstrated that AZT induced p21 up-regulation and inhibition of cell growth in TOV21G cells. Taken together, these data imply that AZT is a potential chemotherapy drug for curing clear-cell-type OC.
We found that up-regulation of p21 caused the suppression of p27 and CHK1 in TOV21G cells. Up-regulation of p21 by DNA damage also has been shown to inhibit CHK1 expression in human retinoblastoma cells (34). Contradicting evidence has indicated that p21 induction is responsible for up-regulation of p27 in ovarian endometrioid adenocarcinoma cells (35). Similar results showed that a significant inverse association existed between p27 and p21 in patients with OC and endometrial cancer (36).The detailed mechanism through which p21 is involved in TOV21G cells remains unexplored.
In CaOV3 cells, AZT induced cell cycle arrest in the S phase by regulating the expression of p16 but not p21. We demonstrated that up-regulation of p16 triggers arrest in the S phase; however, the downstream effectors were not identified in this study. A clinical study has demonstrated that higher p16 expression was found in high-grade serous ovarian carcinoma than in low-grade and borderline tumors, implying that p16 may be involved in the development of neoplasia within the ovary (37). Compared with endometrioid, clear cell, or mucinous ovarian carcinoma, high-grade serous carcinoma tumors exhibit significantly higher p16 immunoreactivity (38). Patients with OC and high p16 expression have been reported to have lower overall survival rates than those with low p16 expression (39). Diverse results also have indicated that in patients with low-grade serous carcinoma, expression of p16 was correlated with higher 5-year survival rates than those in patients without p16 (40). In high-grade serous carcinoma patients, no difference in survival rates was observed between patients with and without p16 expression (41). In this study, we found that p16 expression was absent in the CaOV3 cell line, which were obtained from a high-grade ovarian serous adenocarcinoma. AZT-induced cell cycle arrest in the S phase might occur through the up-regulation of p16. The detailed mechanism remains unclear; however, the low IC50 concentration on CaOV3 makes AZT a potential candidate for application in chemotherapy for OC.
In summary, AZT exhibits multiple characteristics of a chemotherapy drug. A low or medium dose of AZT effectively inhibited the proliferation of serous and clear cell OC. Cell cycle arrest was the primary mechanism of action of AZT. Furthermore, AZT modulated the expression of several cell cycle regulating proteins through a p53-independent pathway. Therefore, AZT may present a valuable candidate in the treatment of OC.
Acknowledgements
This work was supported by the Taichung Veterans General Hospital [grant numbers TCVGH-1076402B].
Footnotes
Authors' Contributions
Conceptualization: Y.-T.H.; Data curation: Y.-T.H.; Formal analysis: Y.-T.H.; Investigation: Y.-T.H.; Methodology: Y.-T.H.; Resources: Y.-T.H.; Validation: Y.-T.H.; Visualization: Y.-T.H.; Writing-original draft: Y.-T.H., J.-J.T.; Writing-review: Y.-T.H., J.-J.T.; Editing: Y.-T.H., J.-J.T.; Funding acquisition: J.-J.T.; Project administration: J.-J.T.; Supervision: J.-J.T.
Conflicts of Interest
No potential conflicts of interest relevant to this article were reported.
- Received June 8, 2020.
- Revision received August 28, 2020.
- Accepted September 2, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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