S-Adenosylmethionine Inhibits the Proliferation of Retinoblastoma Cell Y79, Induces Apoptosis and Cell Cycle Arrest of Y79 Cells by Inhibiting the Wnt2/β-Catenin Pathway
Categoria dell'articolo: Original Article
Pubblicato online: 04 ott 2024
Ricevuto: 24 apr 2024
Accettato: 05 ago 2024
DOI: https://doi.org/10.2478/aite-2024-0020
Parole chiave
© 2024 Mushi Liu et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Retinoblastoma is indeed a serious and challenging condition that affects young children. Retinoblastoma, a malignant tumor mainly impacting the retina, which is the light-sensitive tissue located at the back of the eye. In recent years, various treatment options have emerged, offering alternatives to eye removal. Conservative therapies like chemical volume reduction, transpupillary thermotherapy, cryotherapy, laser photocoagulation, and radiotherapy have shown promise in preserving the eye and vision. Additionally, advancements in genetics, interventions, and traditional Chinese medicine have contributed to the evolving field of retinoblastoma treatment (Yan et al. 2021). In the last 30 years, approximately 42% of patients have had their eyes removed, and 12% have lost their lives as a result of retinoblastoma (Luo et al. 2022). While these advancements are encouraging, it is important to acknowledge that the overall survival rate of retinoblastoma patients remains low (Dimaras et al. 2015). Challenges such as blindness continue to impact patients, highlighting the need for further research, improved treatments, and increased awareness. Medical professionals, researchers, and policymakers need to work together to enhance early detection methods, develop more effective treatments, and offer essential assistance to enhance the prognosis and enhance the quality of life for individuals with retinoblastoma in China and globally (Pascual-Pasto et al. 2019).
Indeed, S-adenosylmethionine (SAM) is a vital biomolecule within mammalian cells. Its significance lies in its involvement in essential metabolic pathways such as transmethylation, transsulfuration, and aminopropylation pathways. SAM has been utilized as a prophylactic medication for the treatment of mood disorders, fibromyalgia, joint pain, intrahepatic cholestasis (Vincenzi et al. 2018), and alcoholic liver disease (Lu et al. 2020). Furthermore, urine SAM levels have been identified as a noninvasive biomarker for monitoring the decline of renal function in the early stages of chronic renal disease (Kruglova et al. 2021). SAM is indeed involved in various cellular processes beyond its main role as a methyl donor. It has great importance in regulating cell proliferation, apoptosis, metastatic invasion, and the cell cycle (Parashar et al. 2015; Hayashi et al. 2018). SAM has demonstrated inhibitory effects on osteosarcoma cells, tumor metastasis, breast cancer (Mahmood et al. 2020), gallbladder cancer (Liu et al. 2020), colon cancer (Zsigrai et al. 2020), and more.
The Wnt/β-catenin signaling pathway is a highly conserved intracellular signal transduction pathway that significantly influences a range of biological processes, including the regulation of cell adhesion, proliferation, apoptosis, and inflammatory responses (Perugorria et al. 2019). Abnormal expression of this pathway has been implicated in tumorigenesis and tumor development (Spitzner et al. 2021). For instance, the activation of the Wnt/β-catenin pathway by AXIN1 has been shown to promote the progression of gastric cancer. In gliomas, dysregulation of this pathway contributes to the clinical malignancy and prognosis of patients (Bai et al. 2017; He et al. 2019). Furthermore, the potential therapeutic targeting of the Wnt/β-catenin pathway by IGF2BP1 and its role as a predictive marker for colon cancer is being actively explored. This could enhance the accuracy of colon cancer diagnosis and potentially reduce the costs associated with detection and treatment (Singh et al. 2023).
We are conducting further experiments to explore the mechanisms by which SAM influences retinoblastoma. Dysregulation of the Wnt/β-catenin signaling pathway, which is recognized for its involvement in cellular proliferation, differentiation, and apoptosis, has been linked to various cancers, including retinoblastoma. We can potentially discover new targets for clinical treatment by investigating whether SAM affects the proliferation, apoptosis, and other cellular processes of retinoblastoma through the Wnt/β-catenin signaling pathway. Furthermore, by studying whether SAM could reduce the expression of the Wnt/β-catenin protein signaling pathway, we can potentially uncover valuable information regarding its therapeutic potential. Overall, our research can establish a novel theoretical foundation for the clinical management of retinoblastoma and contribute to our comprehension of the molecular mechanisms underlying this disease.
All experiments involving mice were approved by the Animal Centre of the Medical College of South China University of Technology (ethical number: 2023105). SAM (Coolaber, China; Dissolved in ultrapure water); HLY78 (HY-122816, Med Chem Express, USA).
Retinoblastoma Y79 (Shanghai Zhongqiao Xinzhou, China,
Y79 (5000 cells per well) were cultured in 96-well plates containing 100 µL of growth medium. Y79 cells were treated with different concentrations of SAM (ranging 0–2.4 mM) for various time points (24 h, 48 h, and 72 h), ARPE-19 (3000 cells per well) cells for 72 h. All wells were incubated with 10 µL CCK8 and incubated continuously at 37°C for 2 h. Absorbance was measured at 450 nm using an ELISA reader. The reported results represent the mean values obtained from at least three independent wells.
Single-cell suspension was prepared from Y79 cells in the logarithmic growth phase and inoculated in 12-well plates. After adding SAM 1 mM and 2 mM for 48 h, 2× of EdU working solution (20 µM), pre-warmed at 37°C, was added in equal volume to each well, and continued to be incubated for 5.5 h at 37°C, 5% CO2 cell culture box. Immunofluorescence staining was performed according to the instructions of BeyoClick™ EdU-594 Cell Proliferation Detection Kit (Beyotime, China), and finally blocked by adding an anti-fluorescence quencher containing DAPI. The cells were observed under a fluorescence microscope (Zeiss, Germany), and three fields of view were randomly selected. The cell proliferation rate was equal to EDU-positive cells/DAP-positive cells. The cell proliferation rate of the three fields was taken as the mean.
Logarithmic growth phase Y79 cells were prepared as single-cell suspension. The cell concentration was adjusted to 1 × 106 cells/mL. Approximately 2–3 drops (about 30–50 µL) of the cell suspension were evenly smeared onto autoclaved glass slides. The slides were air-dried for 10 min at room temperature, followed by washing with phosphate-buffered saline (PBS) for 3 min × 3 times. The cells were fixed in 95% ethanol for 10–15 min, and then washed with PBS 3 times, each for 3 min. Subsequently, the slides were rinsed with deionized water once for 3 min. Staining was carried out using hematoxylin for 3 min, followed by a quick rinse with running water for 3 s. Differentiation was achieved by treating the slides with 1% hydrochloric acid ethanol for 10 s, and then counterstained with eosin for 4 min. Dehydration was performed using a series of ethanol gradients (75%, 80%, 90%, 95%, 95%, 100%, 100%) for 1 min each. The slides were cleared in xylene I and II and mounted with neutral mounting medium. Images were scanned and observed using Digital Pathology Scanner (Leica, Germany). Three random fields were selected. To determine the mitotic index, we calculated the ratio of the number of mitotic cells to the total number of cells.
The samples were quickly and accurately intercepted and then fixed overnight in 2.5% glutaraldehyde. Rinsed with 0.1 M phosphoric acid rinse solution 3 times for 15 min each time. Fixed the samples in 1% osmium acid for 2 h at 4°C in a refrigerator and washed with ultrapure water 3 times for 15 min each time. Gradient dehydration followed by embedding was placed in a constant temperature oven for polymerization, slices (70 mm) were cut using an ultrathin sectioning machine (Leica), and 3% uranyl acetate-lead citrate was double-stained and then visualized under a transmission electron microscope (Hitachi, Japan) and photographed.
Harvested Y79 cells were washed twice with pre-cooled PBS and then fixed in 70% ice-cold ethanol at –20°C overnight. The ethanol was removed by centrifugation and the precipitate was resuspended in 1× PBS. The cells were resuspended in 0.5 mL of Staining Solution, which was prepared by adding 100 µL of RNase A solution and 200 µL of Nuclear Dye to 10 mL of 1× Assay Buffer. The cell suspension was then incubated in the dark at 37°C for 30 min. The cells were washed twice with PBS and resuspended. Cells were analyzed within 48 h on a high-end analytical flow cytometer (BD LSRFortessa Configuration Penta-Laser 18-Colour System, USA) using the appropriate channel (Ex/Em = 535/615 nm), data were analyzed by FlowJO 10.8.1 software (Becton, Dickinson and Company, USA).
In this study, apoptosis was detected using Annexin V-AbFluor 488/PI Double Staining Apoptosis Detection Kit (Abbkine, Wuhan, China). Briefly, Y79 cells and different concentrations (1 mmol/L and 2 mmol/L) of SAM-treated Y79 cells (3 × 105 ↑ cells/well) were grown in 6-well plates.
After 48 h of incubation, the supernatant was discarded, and the cells were rinsed with pre-cooled PBS and then resuspended with 1× Binding buffer. Subsequently, 5 µL of Annexin V-fluorescein isothiocyanate (V-FITC) and 2 µL of propidium iodide (PI) were added to the cell suspension, mixed homogeneously, and incubated at room temperature for 15 min under light. Finally, the apoptosis rate was assessed via flow cytometry within 1 h, in accordance with the instructions provided in the kit.
According to the instructions of Tunel Apoptosis Detection Kit (green fluorescence, Abbkine, Wuhan, China), Y79 (about 1 × 106 cells/mL) was treated with different concentrations (1 mmol/L and 2 mmol/L) of SAM for 48 h. The supernatant was removed, washed twice with PBS, fixed with 4% paraformaldehyde, washed again and permeabilized, and then stained in the dark at 37°C with tunel reagent for 1 h, and photographed with an orthogonal fluorescence microscope (Zeiss) with three randomly selected fields of view. The apoptotic index was equal to Tunel-positive cells/DAPI-positive cells. The apoptotic index of the three fields was taken as the mean.
Pre-treated cellular RNA was extracted using Trizol reagent (TIANGEN, China), and RNA (10 µg) from each sample was reverse transcribed using PrimeScript RT Master Mix (Perfect Real Time) (Takara, Japan). TB Green Premix Ex Tag Il (Tli RNaseH Plus) was used for PCR, and each real-time PCR was performed on a real-time fluorescent quantitative PCR instrument (BIO-RAD, USA) with three replicate wells per group, totaling 25 µL of reaction mixture. Relative expression of Ki67, CASPASE 3, BAX, BCL-2, MMP-2, MMP-9, VEGE, TGF-β, cycD, P21, TP53, Wnt2, β-CATENIN, cycD, c-MYC c-JUN, Axin, and GSK-3β were normalized to GAPDH respectively. The primers were as follows (Table 1):
The primers of RT-PCR
| Genes | Primer sequences |
|---|---|
| F: 5′-ACCCACTCCTCCACCTTTGAC-3′ | |
| R: 5′-TGTTGCTGTAGCCAAATTCGTT-3′ | |
| F: 5′-ACGCCTGGGTTACTATCAAAAGG-3′ | |
| R: 5′-CAGACCCATTTACTTGTGTTGGA-3′ | |
| F: 5′-GAAATTGTGGAATTGATGCGTGA-3′ | |
| R: 5′-CTACAACGATCCCCTCTGAAAAA-3′ | |
| F: 5′-CCCGAGAGGTCTTTTTCCGAG-3′ | |
| R: 5′-CCAGCCCATGATGGTTCTGAT-3′ | |
| F: 5′-GGTGGGGGTCATGTGTGTGTGG-3′ | |
| R: 5′-CGGTTCAGGTACTCAGTCATCC-3′ | |
| F: 5′-CCCACTGCGGTTTTCTCGAAT-3′ | |
| R: 5′-CAAAGGGGGTATCCATCGCCAT-3′ | |
| F: 5′-TGTACCGCTATGGTTACACTCG-3′ | |
| R: 5′-GGCAGGGGACAGTTGCTTCT-3′ | |
| F: 5′-AGGGCAGAATCATCACGAAGT-3′ | |
| R: 5′-AGGGTCTCGATTGGATGGCA-3′ | |
| F: 5′-CTAATGGTGGAAACCCACAACG-3′ | |
| R: 5′-TATCGCCAGGAATTGTTGCTG-3′ | |
| F: 5′-CGATGGAACTTCGACTTTGTCA-3′ | |
| R: 5′-GCACAAGGGTACAAGACAGTG-3′ | |
| F: 5′-GAGGTTGGGCTCTCTGACTGTACC-3′ | |
| R: 5′-TCCGTCCCAGTAGATTACCAC-3 | |
| F: 5′-CCTGTTCCCCTGAGGGTATT-3′ | |
| R: 5′-CCATCAAATCAGCTTGAGTAGCC-3 | |
| F: 5′-GCCTTTGTTTATGCCATCTCCT-3′ | |
| R: 5′-CTTGGCGCTTCCCATCTTCTT-3′ | |
| F: 5′-TGCACCCACATCATCTACAG-3′ | |
| R: 5′-ACTCGTCATTCCACTCCCAT-3′ | |
| F: 5′-AGTGAGCTCAGGAGGAGGTGGTGTAA-3′ | |
| R: 5′-AGTAAGCTTGTGAGGGCAGAGGTGTC-3′ | |
| F: 5′-TCCAAGTGCCGAAAAAGGAAG-3′ | |
| R: 5′-CGAGTTCTGAGCTTTCAAGGT-3′ | |
| F: 5′-GACAAGATCGCAGAGGAAGG-3′ | |
| R: 5′-ACCCCCACAGTCAAACTCGTC-3′ | |
| F: 5′-CCTGGGAACTCCAACAAGGG-3′ | |
| R: 5′-GGGGTCGGAAGACCTTAGTC-3′ |
Y79 cellular proteins were extracted in a mixture of RIPA lysate (Beyotime) and 2% phosphatase protease (Beyotime) for 30 min on ice. The extracts were centrifuged at 14,000×
To assess the impact of SAM on the growth of Y79 cells, a retinoblastoma xenograft model was established with 18 female BALB/c nude mice, aged 6 weeks, which were obtained from the Experimental Animal Centre of South China University of Technology. A Y79 cell suspension (8 × 106 cells) mixed with a 1:1 ratio of PBS and matrix gel was injected into the right side of the posterior flank of each mouse. Tumors were allowed to grow until reaching a volume of 200–500 mm3, at which point they were randomly allocated into three groups, with each group comprising six mice: (1) Control-0mM (0 mM SAM: sterile physiological saline), (2) SAM-1mM (40 mg/kg), (3) SAM-2mM (80 mg/kg). The mice in the SAM-treated groups received intraperitoneal injections of SAM (at either 40 mg/kg or 80 mg/kg) or an equivalent volume of saline 5 times, with a 2-day interval between each administration. During the experiment, the tumor volumes (calculated using the formula: Volume = (length × width2)/2) and the body weights of the mice were measured at regular intervals. After the final administration (1 day after tumor cell injection), the mice were euthanized, and the tumors were collected and weighed. All animal experiments were conducted according to the guidelines established by the Laboratory Animal Care and Use Committee of the School of Medicine, South China University of Technology.
The exfoliated tumors were fixed with 4% paraformaldehyde, embedded in paraffin, processed into tissue blocks, and sectioned into 4 µm-thick slices. These sections were then subjected to immunohistochemical analysis for cell proliferation marker Ki67 (27309-1-AP, 1:100, Proteintech) using the Rabbit Secondary Anti HRP Immunohistochemistry Kit (Zhongshan Jinqiao, China), following the kit’s instructions.
All experiments were repeated at least 3 times. Data are presented as mean ± standard deviation from three independent experiments. Differences between three or more groups were analyzed using one-way analysis of variance (ANOVA). GraphPad Prism 9 (GraphPad, USA) was used to analyze statistical significance.
Cancer cells are characterized by uncontrolled tumor cell growth resulting from dysregulated cell proliferation. Targeted containment of cell proliferation has been a significant focus in tumor therapeutics. Therefore, we first investigated the effect of SAM on the viability of Y79. First, Y79 cells were treated with different concentrations of SAM for 24 h, 48 h, and 72 h. The results showed that SAM was able to inhibit the viability of Y79 cells in a dose-dependent and time-dependent manner, and the IC50 of Y79 cells after 48 h of treatment was 1.713. The data presented in Figure 1a demonstrate that SAM exhibited a dose-dependent and time-dependent reduction in the viability of Y79 cells (
SAM inhibited cell viability and proliferation of Y79 cells.
In order to investigate the effect of SAM on the morphology of retinoblastoma, first, in H&E staining, it was observed that the ratio of nuclear division of Y79 cells in the field decreased with the increase of SAM concentration (Figures 2a–c;
SAM affects the morphology of retinoblastoma cell lines and their ultrastructure.
Apoptosis of Y79 cells was quantified using flow cytometry by detecting apoptosis by the membrane-linked protein V-FITC/PI binding assay after SAM treatment for 48 h. After treatment with 1 mM and 2 mM SAM, the apoptosis rate observed in the experimental groups was significantly higher compared to the control group (3.16%;
SAM inducing apoptosis and cell cycle arrest of Y79 cells were treated with SAM (1 mM, 2 mM) for 48 h.
TUNEL assay results revealed a greater dose-dependent increase in the rate of apoptosis in the SAM-treated group compared to the control group, which exhibited typical apoptotic features (Figures 3b–e;
To understand more precisely the specific time points of the cell cycle in which SAM is mainly involved, we used flow cytometry to study its cell cycle distribution. Our results showed that the SAM-treated group exhibited a higher proportion of Y79 cells in the G1 phase (Y79: 48.70%–57.80%;
To explore the mechanism by which SAM acts on Y79 cells, RT-PCR and Western blot analyses were conducted. RT-PCR was shown that it’s lower for cell proliferation gene Ki67, metastasis-related genes mmp-2, mmp-9, cell cycle-related genes P21, TP53, vascular endothelial factor (VEGF), transforming growth factor (TGF)-β compared to control group, while apoptosis-related genes caspase3, bax, bcl-2 were higher (Figures 4b and c,
SAM treatment downregulated the expression of genes associated with the Wnt2/β-catenin signaling pathway in Y79 cells. Y79 cells were treated with SAM (1 mM, 2 mM) for 48 h.
To further show that SAM may exert its oncostatic effects on retinoblastoma through the Wnt2/β-catenin signaling pathway, we used the Wnt2/β-catenin agonist, HLY78 (1 µM), which acted on retinoblastoma cell Y79 compared with control-0mM-SAM-1mM and SAM-2mM group. Four groups: (1) control-0mM; (2) HLY78; (3) AM-1mM + HLY78; (4) SAM-2mM + HLY78 were detected to be expressed at the level of cell proliferation, related genes’ mRNAs and proteins. The results showed that the addition of agonist HLY78 treatment significantly promoted cell proliferation (Figure 5a;
SAM attenuated the cancer-promoting effects induced by activation of the Wnt2/β-catenin signaling pathway (concentration-dependent attenuation).
To further confirm
SAM dampened cell proliferation of Y79 cells in vivo.
Retinoblastoma is the predominant primary intraocular tumor in kids and its low incidence still causes significant distress for affected families. Current treatments often yield unsatisfactory outcomes, largely due to the high risk of children losing their eyes to the tumor. In contrast to conventional radiotherapy, SAM, a natural compound found in the human body and commonly used as a nutritional supplement, has shown promise in clinical use for treating conditions such as depression, joint disorders, fibromyalgia, liver disease, and migraines (Galizia et al. 2016). Given its natural origin and minimal adverse effects, SAM exhibits potent anti-tumor activity and has emerged as a potential new anti-cancer drug for children with retinoblastoma. As awareness of retinoblastoma grows, an increasing number of drugs are being discovered and researched for their efficacy in clinical settings. Our findings demonstrate that SAM inhibits retinoblastoma development in a dose-dependent and time-dependent manner. Further investigation into its mechanism of action revealed its potential in attenuating the Wnt2/β-catenin signaling pathway, suggesting that SAM holds promise for using as a therapeutic agent for retinoblastoma.
This study aimed to explore the mechanism underlying the impact of SAM on the proliferation, apoptosis, and cell cycle of retinoblastoma. SAM has been demonstrated to be an effective therapeutic approach for diverse conditions, including depression and liver disease, without causing serious side effects, unlike common chemotherapeutic drugs. Therefore, SAM has the potential to become a new anti-tumor drug. It is well-known that SAM has an antiproliferative effect. The first step in this study was to determine the optimal effective concentration of SAM against the retinoblastoma cell line Y79, which was found to be 1–2 mM after a 48-h treatment. Flow cytometry analysis revealed a significant reduction of cancer cells in the S phase and an accumulation of cells in the G1 phase among the treated cells. Targeting the cell cycle machinery has shown promise in cancer therapy. In recent years, the Wnt2/β-catenin pathway has been activated in various types of tumor cells, contributing to cell proliferation, apoptosis, differentiation, metastasis, and infiltration. Inhibiting the activated Wnt2/β-catenin signaling pathway can lead to programmed cell death and growth inhibition in various tumor cells. Based on these findings, the Wnt2/β-catenin pathway may serve as a promising marker and therapeutic target for different types of malignancies. It is worth noting that our results demonstrated that SAM significantly inhibited the protein levels of Wnt2 and β-catenin, and decreased the mRNA expression of GSK-3β and AXIN1, which are major downstream factors associated with the Wnt2/β-catenin signaling pathway. Our RT-PCR analysis demonstrated that SAM reduced the mRNA expression levels of Wnt2/β-catenin and its downstream target genes, including C-MYC, C-JUN, and cycD. Additionally, SAM decreased the expression levels of cell cycle-related genes P21 and TP53, as well as vascular endothelial factors VEGF and TGF-β. This further supports the notion that SAM may play an anticancer role in retinoblastoma by inhibiting the Wnt2/β-catenin signaling pathway. Interestingly, there were statistically significant differences between the SAM-1mM and SAM-2mM groups compared to the control-0mM group, but there were no significant differences in some genes (e.g., Wnt2, β-catenin, cycD, c-JUN, c-MYC, bcl2, mmp2, mmp9, Ki67, etc.) between the SAM-1mM and SAM-2mM groups, which suggests that by reaching the dose administered to the low dose group, (it seems) the saturating administered dose is reached, so further increase in the administered dose does not increase the actual effect of the administered dose. Furthermore, in our experimental study using nude mice, we observed a significant reduction in tumor weight and volume during the course of SAM treatment. Biochemical indicators related to blood from the eyeballs of nude mice after tumor excision indicated that the drug does not cause liver or kidney toxicity. Immunohistochemical analysis also indicated a reduction in the expression levels of Ki67 after SAM treatment, which is consistent with our