Abstract
Background/Aim: Stem-like cancer cells are believed to be the leading cause of therapy resistance in malignant melanoma (MM). All-trans retinoic acid (ATRA) differentiation therapy is considered a promising approach to eradicate stem-like cancer cells, but some melanoma cells are resistant to ATRA. This study aimed to examine whether resveratrol (RS), a natural polyphenol compound, could improve the response of MM stem-like cells to ATRA and explore the possible underlying mechanisms. Materials and Methods: MM stem-like cells were established from a spheroid model of A375 human MM cell line. The response to RES alone and in combination with ATRA, was examined through analysis of cancer stemness, cell viability, and protein expression. Results: The stem-like cells showed resistance to the anticancer drug docetaxel; however, the combination of RES and ATRA augmented the effects of docetaxel. Accordingly, these combinatorial effects were associated with significant inhibition of the expression levels of stemness markers CD133, OCT4, CD271, and ABCG2. The tested combinations also led to a significant increase in melanocyte differentiation marker SOX9, while efficiently suppressing the dedifferentiation marker SOX10. Notably, RES alone effectively up-regulated retinoic acid receptor beta (RARβ) expression and down-regulated crucial mediators like DNMT1, polycomb-group proteins EZH2, and BMI-1, which mechanistically explain how RES enhanced the differentiation-inducing effects of ATRA. Conclusion: The resistance of MM stem-like cells to ATRA can be attenuated by RES and combined applications of ATRA and RES provide a promising strategy for MM treatment.
Malignant melanoma (MM) is a highly aggressive melanocytic neoplasm and one of the most fatal forms of skin cancer. Even though it constitutes only 5% of skin cancers, its rapid recurrence, resistance to cytotoxic agents, and fast metastasis leads to the low survival rates of patients (1, 2). Treatments for MM have obtained few satisfactory results; surgery, chemotherapy, and immunotherapy are the most common methods for treatment. Chemotherapy plays an important role in the collective treatment of MM but has limited success because of fast development of drug resistance (2, 3). Melanoma exhibits a high degree of heterogeneity, enabling its progression and therapy resistance (4, 5), and mediated through phenotype switching; allowing a transition in its transcription profile between differentiated/non-cancer stem like cells (non-CSC) and undifferentiated/cancer stem-like cells (CSC) (5). CSCs are known to be responsible for tumor stemness, metastasis, and therapy resistance. Furthermore, the presence of CSCs leads to cancer recurrence and therefore their complete eradication can have major therapeutic benefits (5-7). Several studies have indicated that highly aggressive cancer cells appear to be relatively undifferentiated or dedifferentiated. Induced differentiation of stem-like cells into a more chemo-susceptible phenotype has proven to be effective in the treatment of many types of human malignancies (8). Therefore, treatments that effectively target melanoma CSCs by promoting melanoma CSC differentiation towards a non-CSC state could lead to better therapeutic options.
All-trans retinoic acid (ATRA), a derivative of vitamin A retinoids, represents one of the most frequently used and clinically effective differentiation therapies for acute promyelocytic leukemia (APL), a stem cell malignancy (9, 10). The use of ATRA as an adjuvant has been shown to be also effective for the treatment of APL and other hematological diseases (10). In vitro and in vivo differential effects of ATRA were also seen in several solid tumors such as melanoma (11, 12). Induction of differentiation by ATRA can increase sensitivity to therapies and reduce stemness capability and tumorigenicity (11-13). However, in the most aggressive phenotypes, retinoic acid resistance can develop, ultimately leading to ineffectual treatment (13, 14). ATRA slows growth and induces differentiation in cancer cells through the activation of retinoic acid receptors (RARs) and retinoid X receptor (RXR) that regulates transcription factors implicated in cellular differentiation (10, 15). It has been suggested that the lessened efficacy of ATRA differentiation activity in non-APL and solid tumors may be associated with aberrant epigenetic mediated silencing of retinoic acid receptor beta (RARβ) (15). The lack of RARβ expression has been observed in a number of malignant tumors especially in MM, where the frequency of loss of expression of RARβ is as high as 70% (16). Furthermore, RARβ depletion promotes tumorigenicity and enhances CSC stemness in a variety of cancers (17). Hence, the status of RARβ expression is considered a critical element in determining ATRA sensitivity in cancer cells. Restoration of the lost expression of RARβ in melanoma may amplify ATRA responsiveness and could be an effective strategy in eliminating CSCs.
Resveratrol (RES) is a naturally occurring polyphenolic compound possessing multifaceted anti-cancer capabilities against various types of cancer including melanoma (18, 19). RES has been shown to enhance the therapeutic potential of anticancer drugs and to sensitize various resistant cancer cells to chemotherapy, making it an appealing anticancer agent, particularly for solid tumors (18-20). Increasing evidence in many studies has also suggested that resveratrol regulates epigenetic machinery by altering gene methylation patterns in a variety of cancer cells, including those with ATRA resistance (20, 21). The demethylating capacity of RES in combined therapy with anti-cancer drugs effectively reverses the process of carcinogenesis by inducing redifferentiation and apoptosis (18, 20, 22). Based on the findings that epigenetic aberrations in MM that lead to silenced RARβ are also responsible for the resistance to ATRA and promotion of tumorigenesis. We therefore hypothesized that epigenetic regulatory effects of RES could potentially improve transcriptional regulation that underlies ATRA resistance in melanoma. In this context, the present study was undertaken to assess these possible actions. Using MM stem-like cells established from a spheroid model of ATRA resistant human MM cells, we aimed to clarify whether the resistance of MM stem-like cells to the differentiation-inducing effect of ATRA could be attenuated by RES, and if its effect could help overcome the chemoresistance of MM stem-like cells.
Materials and Methods
Materials. All cultures and reagents were purchased from Nacalai tesque (Kyoto, Japan) and Pepro Tech (Cranbury, NJ, USA) unless otherwise stated. Fetal bovine serum (FBS) was purchased from BioWest (Nuaillé, France). Docetaxel was from TCI (Tokyo, Japan), and RES and ATRA were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan). EPZ-6438 (EZH2 inhibitor) and PTC-209 (BMI-1 inhibitor) were purchased from Selleck (Houston, TX, USA) and from MedChem Express (Monmouth Junction, NJ, USA), respectively. PCR primers were from Japan Gene Research Institute (Miyagi, Japan).
Cell culture and isolation of MM stem-like cells. Human skin-derived MM cell line A375 from ECACC (London, UK) was used. The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 0.5% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO2. To isolate MM stem-like cells, we utilized the tumor-sphere forming capacity of the cells in a three-dimensional (3D) culture system using an ultra-low attachment 96-well U plate (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) (23). The cells cultured in a two-dimensional culture system were washed with Ca2+-Mg2+-free phosphate-buffered saline (PBS), treated with 0.5 g/l-trypsin/0.53 mmol/l-EDTA solution, and resuspended in stem cell medium consisting of DMEM/Ham’s F12 (Wako, Osaka, Japan), 2% B-27 (Gibco, Tokyo, Japan), 20 ng/ml epidermal growth factor (EGF), and 20 ng/ml basic fibroblast growth factor (bFGF). The cells were suspended in the stem cell medium at a density of 5,000 cells/ml, transferred to each well of the ultra-low attachment 96-well U plate, and cultured at 37°C in a humidified atmosphere with 5% CO2 for seven days.
MTT assay. For assessing the viability of cells, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) was used (24). MTT was dissolved in PBS (5 mg/ml), and the MTT solution was diluted in PBS to gain the final MTT solution (0.25 mg/ml) for MTT assay. The supernatant of the cells treated with each reagent was removed and collected. The collected cells were suspended in 250 μl of MTT solution and incubated for 1 h at 37°C and 5% CO2. Thereafter, the cell suspension was centrifuged at 800 × g or 3 min at 20°C to remove the MTT solution, and then dissolved in 250 μl of dimethyl sulfoxide (DMSO). Absorbance measurements were performed at 540 nm with a microplate reader (SUNRISE Rainbow RC-R, Tecan Japan, Kanagawa, Japan).
QRT-real time PCR. Total RNA was isolated from cultured cells using the Tissue Total RNA Extraction Mini Kit (Favorgen Biotech Corp., Ping-Tung, Taiwan, ROC). Total RNA (300 ng of each sample) was used for cDNA synthesis using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo, Osaka, Japan). cDNA templates were analyzed by real-time PCR using an ABI Prism 7000 sequence detection system (Applied Biosystems Japan Ltd., Tokyo, Japan) and Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, PR China) according to the following program, 95°C for 10 s, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Primer sets are shown in Table I. Gene expression data were normalized to the expression of the reference gene ribosomal protein L32 (RPL32).
Primer sequences.
Statistical analysis. Differences among groups were analyzed using one-way ANOVA followed by the Tukey-Kramer test, and differences between two groups were analyzed using one-way ANOVA followed by Student’s t-test. All statistical analyses were performed using Ekuseru-Toukei software (Social Survey Research Information Co., Ltd., Tokyo, Japan). Differences with p-values of 0.05 or less were considered statistically significant. All experiments were conducted with a minimum of three samples from three independent experiments and the data are expressed as the mean±standard deviation (S.D.). The number of samples for each experiment is shown in the respective figure legend.
Results
Effect of combined use of RES and ATRA on MM stem-like cells. We examined whether RES could enhance the differentiation-inducing effect of TRA on MM stem-like cells that are resistant to differentiation induction by ATRA. After treatment with 25 μM RES for 24 h and subsequent 20 μM ATRA for 24 h, the mRNA levels of CSC markers decreased in all cells compared to the control group, including prominin 1 (CD133), octamer-binding transcription factor 4 (OCT4), nerve growth factor receptor (CD271), ATP binding cassette transporter G2 (ABCG2) (7, 25-27). The difference was significant (p<0.05, p<0.01) (Figure 1A). This treatment combination with RES and ATRA was selected as the treatment with the maximum effect among the treatments examined. In addition, the mRNA level of the differentiation marker sex determining region Y-box (SOX)9, which shows an inverse correlation as a marker related to the differentiation of melanocytes and MM (28), was significantly increased (p<0.01) in the RES/ATRA combination treatment group compared to the control group (Figure 1B). The mRNA level of the dedifferentiation marker SOX10 (29) was significantly decreased (p<0.05) in the RES/ATRA combination treatment group compared to the control group (Figure 1C).
Combined treatment with resveratrol (RES) and all-trans retinoic acid (ATRA) suppressed cancer stem cell markers and induced differentiation markers in malignant melanoma stem-like cells. A375 cells were three-dimensionally cultured for one week in an ultra-low adhesion 96-well U plate to form a spheroid. After treating the spheroid with RES (25 μM) for 24 h, ATRA (20 μM) was added to the same well. At 24 h after the treatment, mRNA expression was analyzed. (A) Cancer stem markers. (B) Differentiation marker (SOX9). (C) Dedifferentiation marker (SOX10). Data are mean±SEM, n=3. *p<0.05 and **p<0.01.
Combination effect of RES and ATRA on anticancer drug resistance in MM stem-like cells. Chemotherapy and radiation therapy are effective against proliferating cells, so CSCs that maintain a dormant state and divide slowly are resistant to them (30). If we assume that the combination of RES and ATRA causes cancer stemness to decrease and differentiation to be induced, and then sensitivity to cell cycle-dependent anticancer drugs may be restored. After the combination treatment of RES and ATRA, the cells were treated with 1,000 nM of the anticancer drug Docetaxel and the cell viability was determined. As shown in Figure 2, the cell viability was significantly (p<0.05) lower in the RES/ATRA/Docetaxel combination treatment group compared to either the control group or the ATRA/Docetaxel combination treatment group.
The combination of resveratrol (RES) and all-trans retinoic acid (ATRA) reduced the viability of melanoma stem-like cells induced by Docetaxel. A375 cells were three-dimensionally cultured for one week in an ultra-low adhesion 96-well U plate to form a spheroid. After treating the spheroid with RES (25 μM) for 24 h, ATRA (20 μM) was added to the same well. At 24 h after this treatment, Docetaxel (1000 nM) was added for further 24 h, and cell viability was measured using MTT assay. Data are mean±SEM, n=5. *p<0.05 and **p<0.01.
Effect of RES on the expression of RARβ and DNA methyltransferase (DNMT)1. The expression of ATRA receptor RARβ is suppressed in malignant melanoma, and this suppression has been found to correlate with the loss of ATRA-dependent cell differentiation-inducing effect (31). Furthermore, abnormal DNA methylation of RARβ is thought to be the cause of its suppressed expression in MM (32). Therefore, since it seems that the expression of RARβ will be restored by alleviating the abnormal DNA methylation state, we investigated changes in the expression of the ATRA receptor RARβ and DNMT1 due to RES treatment. We found that RARβ mRNA expression was significantly (p<0.05, p<0.01) increased by the RES treatment in a time-dependent manner (Figure 3A), and DNMT1 mRNA expression was decreased in a time-dependent manner by the treatment. A significant (p<0.01) change was confirmed in the 48-hour RES treatment compared to the 0-h RES treatment (Figure 3B).
Resveratrol (RES) treatment increased the expression level of an all-trans retinoic acid (ATRA) receptor, RARβ, and decreased the expression level of a DNA methyltransferase, DNMT1, in malignant melanoma stem-like cells in a time-dependent manner. A375 cells were three-dimensionally cultured for one week in an ultra-low adhesion 96-well U plate to form a spheroid. The spheroids were treated with RES (25 μM) for 24 and 48 h, and mRNA expression was analyzed every 24 hourS. (A) RARβ. (B) DNMT1. Data are mean±SEM, n=3. *p<0.05 and **p<0.01.
Effect of RES on the expression of ATRA-regulated transcriptional regulators EZH2 and BMI-1. After binding to the nuclear receptor RARβ, ATRA triggers the transcription of genes that control differentiation and apoptosis (31). However, ATRA resistance is induced by up-regulation of factors that control the expression of these target genes. Therefore, we investigated the transcriptional regulators EZH2 and BMI-1, which are involved in RES-mediated transcriptional repression of target genes. As a result, RES treatment decreased the mRNA expression of EZH2 and BMI-1 in a time-dependent manner, and the difference was significant (p<0.01) in the RES 48-hour treatment compared to the RES 0-hour treatment for both genes (Figure 4).
Resveratrol (RES) treatment suppressed the expression levels of all-trans retinoic acid (ATRA) transcription regulators EZH2 and BMI-1 in malignant melanoma stem-like cells. A375 cells were three-dimensionally cultured for one week in an ultra-low adhesion 96-well U plate to form a spheroid. The spheroids were treated with RES (25 μM) for 24 and 48 h, and mRNA expression was analyzed every 24 h. (A) EZH2. (B) BMI-1. Data are mean±SEM, n=3. *p<0.05 and **p<0.01.
Effect of RES on the expression of target genes of EZH2, BMI-1, and ATRA. As shown in Figure 4, inhibition of EZH2 and BMI-1 by RES was confirmed. In order to further ascertain the inhibitory effect of RES, we examined the effect of RES on the expression of cyclin dependent kinase inhibitor 2A (P16), cyclin dependent kinase inhibitor 2D (P19) and cyclin dependent kinase inhibitor 1A (P21), which are negatively regulated by EZH2 and BMI-1 (33-36). We found that the expression of each mRNA was significantly increased (p<0.01 and p<0.05) (Figure 5A). In addition, since these target genes are also up-regulated by ATRA, we examined the changes when RES and ATRA were treated together based on the hypothesis that a greater increase in the expression would be observed when ATRA is used in the combination. A more significant increase in expression was confirmed by the combination of RES and ATRA than by RES alone (Figure 5B).
Treatment with resveratrol (RES) alone or in combination with RES and all-trans retinoic acid (ATRA) increased the expression levels of P16, P19, and P21, which are target genes of EZH2 and BMI-1, in malignant melanoma stem-like cells. A375 cells were three-dimensionally cultured for one week in an ultra-low adhesion 96-well U plate to form a spheroid. After treating the spheroid with RES (25 μM) for 24 h, ATRA (20 μM) or vehicle was added to the same well. At 24 and 48 h after this treatment, mRNA expression was analyzed. (A) RES alone (B) RES/ATRA combination. Data are mean±SEM, n=3. *p<0.05 and **p<0.01.
Effects of EZH2 and BMI1 inhibition on the expressions in ATRA target genes. In order to examine whether the effect of RES observed in Figure 5 could be a phenomenon occurring through the inhibition of EZH2 and BMI-1, we used specific inhibitors of EZH2 and BMI-1 to determine the effects on the expressions in ATRA target genes. We found that the expression of P21 mRNA increased in all groups compared to control group, and there was a significant difference between the control group, EPZ-6438 treatment group, or PTC-209 treatment group and the combination treatment group (p<0.01). The mRNA expression of P16 and P19 increased significantly (p<0.05, p<0.01) in the EPZ-6438 treatment group and the combination treatment group. Also, the expression level showed a tendency to decrease in the PTC-209 treatment group in comparison with control group. The rate of increase in the expression of P21, P16, and P19 in the combination treatment group showed a similar tendency in the changes caused by RES (Figure 6).
Treatment with EZH2 inhibitor and BMI-1 inhibitor increased the expression of their target genes in malignant melanoma stem-like cells. A375 cells were three-dimensionally cultured for one week in an ultra-low adhesion 96-well U plate to form a spheroid. The spheroids were treated with EPZ-6438 (2 μM), PTC-209 (0.5 μM), and combination of EPZ-6438 and PTC-209 for 48 h, and after that, mRNA expression was analyzed. Data are mean±SEM, n=3. *p<0.05 and **p<0.01.
Discussion
CSCs play an important role in tumor initiation and growth because of their undifferentiated state and capability for self-renewal and multipotency to assemble tumor tissue into a heterogeneous population (37-39). They also contribute to tumor resistance to conventional treatments, and can lead to post-treatment recurrence, progression, and metastasis (40-42). In particular, MM has a high metastatic potential and problematic post metastasis treatment resistance (43). Therefore, CSC regulation in MM is thought to be capable of suppressing cancer recurrence and metastasis, leading to a fundamental treatment solution. In this study, we focused on RES as an agent that has the potential to overcome the resistance of MM stem-like cells to differentiation induction by ATRA. RES, a type of natural polyphenol found in more than 70 plant species, such as red wine, grapes, and peanuts, has antiproliferative and proapoptotic effects, and promotes cell cycle arrest, while it also has anti-metastatic and anti-invasive properties (44, 45). RES can target CSCs both alone or in combination with other compounds and drugs (46).
MM stem-like cells enriched by three-dimensional culture show increased expression of several CSC markers such as OCT4. A decrease in the expression of all markers was observed in the RES/ASTRA combination group; in particular, the expression of OCT4 (47, 48), which induces dedifferentiation and contributes to the acquisition of cancer stemness, was significantly decreased, suggesting that differentiation and decline in cancer stemness may be induced by the combination. Furthermore, CD271 mRNA expression declined to about one-fifth or less of that in the control group. It has been revealed that CD271-positive cells exhibit high tumorigenic and metastatic potential in MM (49), and therefore the significant decrease in CD271 expression in the treatment group could also indicate a decrease in these properties. CSCs have high tumor-forming potential and cannot be eradicated by the present conventional treatments, and their presence causes metastasis and affects cancer recurrence and malignancy (50). Overall, it is considered that there is a commensurate decrease in CSC marker expression and cancer stemness due to the combination of RES and ATRA. Additionally, SOX9 is expressed in cells derived from neural crest cells, including melanocytes, and expression is said to decrease in the order of melanocytes, nevi, primary malignant melanoma, and metastatic MM (51). Conversely, SOX10, a dedifferentiation marker, increases in expression during the progression of malignant melanoma. Previous studies have reported that SOX9 over-expression induces a decreased expression of SOX10 in MM, and that the antiproliferative effect of SOX10 deficiency is due to increased expression of SOX9 (52). Thus, their expression shows an inverse correlation, and in this study, such a correlation was confirmed between SOX9 and SOX10, suggesting that the combination of RES and ATRA might suppress dedifferentiation and induce differentiation in the cells from tumor spheroids. As described above, it is confirmed that the combined use of RES and ATRA on MM stem-like cells enriched by the three-dimensional culture method reduces malignancy and induces differentiation, alleviating resistance to anticancer drugs.
Since it was revealed that the combination of RES and ATRA could induce the differentiation of MM stem-like cells, we next investigated the mechanism by which RES alleviates ATRA resistance based on a decrease in ATRA receptor expression and high expression of the ATRA transcriptional regulator. It has been reported that BMS493, a pan-ATRA receptor antagonist, blocks ATRA-induced apoptotic index in cancer cells (53), indicating that the presence of ATRA receptor is essential for the action of ATRA. Among the ATRA receptors, the expression of RARβ is subject to epigenetic modification (54). In addition, the expression of DNMT1 in MM increases in the order of nevus, primary malignant melanoma, and metastatic malignant melanoma, and is increased in undifferentiated cells and is involved in maintaining cell proliferation and suppressing differentiation (55). Therefore, the decreased expression of DNMT1 observed in this experiment suggests that the undifferentiated state of MM stem-like cells may be alleviated. Also, it has been reported that RES can reduce the enzymatic activity and mRNA expression level of DNA methyltransferase in breast cancer cells (56), so inhibition of DNA methylation by RES restores the expression of hypermethylated RARβ.
PcG proteins form complexes and act on target genes, aggregating chromatin, inhibiting the elongation reaction of RNA polymerase, and controlling transcription. This mechanism involves the sequential action of two repressor complexes, polycomb repressive complex (PRC)2 and PRC1. The catalytic subunit of PRC2, EZH2 catalyzes the methylation of H3 histone of H3K27Me3, which inhibits the transcription of target genes PRC1 then binds to histone H3K27me3 via chromobox, a component of PRC1, and the RING 1B protein, which is optimally activated upon binding to BMI-1, and modifies histone H2AK119ub1, causing nucleosome aggregation and transcription suppression (57). In particular, EZH2 has been reported as a high-grade marker for malignant tumors that occur in the prostate and breast, and its over-expression is closely related to malignant transformation of cancer (58). The expression of EZH2 and BMI-1 in MM increases with the malignant transformation of MM that metastasizes from the nevus (59, 60). Thus, the reduction in the expression of EZH2 and BMl-1 observed in this study clearly indicated the suppression of malignancy in the spheroid-derived MM stem-like cells. Also, EZH2 suppresses RAR signal transduction, inhibiting ATRA target gene expression by interacting with preferentially expressed antigen in melanoma, PRAME (61), and the suppression is maintained in a BMI-1-dependent manner by interacting with PRC1 (62). Therefore, the continuous action of EZH2 and BMI-1 suppresses RAR signals and transcription of target genes. In this study, we confirmed that RES decreased the expression of EZH2 and BMI-1, and increased the expression of P21, P16, and P19, which are transcriptional target genes of EZH2 and BMI-1. This observation suggested that inhibition of EZH2 and BMI-1 might lead to the suppression of ATRA transcriptional target genes. By using EZH2 and BMI-1 specific inhibitors (EPZ-6438 and PTC-209), the increase in the expression of P21, P16, and P19 in the group treated with the combination of EPZ-6438 and PTC-209 showed a similar tendency to the changes caused by RES. Therefore, it was considered that the expression of ATRA transcriptional target genes could be increased through inhibition of EZH2 and BMI-1. These results show that the combination of RES and ATRA might be a new differentiation-inducing therapy for MM.
Conclusion
In conclusion, by restoring ATRA receptor expression and inhibiting ATRA transcriptional regulators through RES treatment, the combined use of RES and ATRA induces differentiation of MM stem-like cells and increases their sensitivity to anticancer drugs. Thus, the combination of ATRA and RES provides a promising strategy for the treatment of MM.
Acknowledgements
This work was completely supported by Inoue Enryou Memorial Foundation of Toyo University, Tokyo, Japan.
Footnotes
Authors’ Contributions
MK performed all experiments of the present study and wrote the manuscript. AS and MO reviewed the manuscript. NV and TY provided the interpretation of experimental results and edited the manuscript.
Conflicts of Interest
The Authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
- Received August 28, 2024.
- Revision received November 7, 2024.
- Accepted November 8, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
