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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

Open Access
|Oct 2024

Figures & Tables

Fig 1.

SAM inhibited cell viability and proliferation of Y79 cells. (A) CCK8 assay was conducted to test the effect of SAM on Y79 cell viability. (B) CCK8 assay was conducted to test the effect of SAM on ARPE-19 cell viability. (C and D) EDU experiment was used to verify the effect of SAM (1 mM, 2 mM) on cell proliferation. (E) The molecular structure of SAM was shown. Each experiment was repeated 3 times independently, and the data are presented as mean ± standard deviation. Data in panels were analyzed using one-way ANOVA, nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). ANOVA, analysis of variance; CCK8, Cell counting kit-8; EDU, 5-Ethynyl-2′-deoxyuridine; SAM, S-adenosylmethionine.
SAM inhibited cell viability and proliferation of Y79 cells. (A) CCK8 assay was conducted to test the effect of SAM on Y79 cell viability. (B) CCK8 assay was conducted to test the effect of SAM on ARPE-19 cell viability. (C and D) EDU experiment was used to verify the effect of SAM (1 mM, 2 mM) on cell proliferation. (E) The molecular structure of SAM was shown. Each experiment was repeated 3 times independently, and the data are presented as mean ± standard deviation. Data in panels were analyzed using one-way ANOVA, nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). ANOVA, analysis of variance; CCK8, Cell counting kit-8; EDU, 5-Ethynyl-2′-deoxyuridine; SAM, S-adenosylmethionine.

Fig 2.

SAM affects the morphology of retinoblastoma cell lines and their ultrastructure. (A–C) Effect of SAM on the morphology of Y79 cells, with a concentration-dependent decrease in nuclear schizophrenic images. (b): Electron microscopic changes in the ultrastructure of retinoblastoma with changes in SAM concentration (12,000×). Control-0mM (a and b): Normal structure of retinoblastoma Y79 cells under transmission electron microscopy. SAM-1mM (Low-dose treatment group: [c and d]): Ultrastructural changes in retinoblastoma Y79 cells. SAM-2mM (high-dose treatment group: [e and f]): Ultrastructural changes in retinoblastoma Y79 cells. Red arrows indicate nuclear grooves, blue arrows denote normal mitochondria, orange arrows represent normal endoplasmic reticulum, yellow arrows point to apoptotic bodies, green arrows indicate swollen, ruptured, vacuolated mitochondria, and purple arrows denote swollen endoplasmic reticulum. Each experiment was repeated for 3 times independently, and the data are presented as mean ± standard deviation. Data in panels were analyzed using one-way ANOVA, nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 (vs. control or low dose administration group). ANOVA, analysis of variance; H&E, Hematoxylin and eosin stain; SAM, S-adenosylmethionine.
SAM affects the morphology of retinoblastoma cell lines and their ultrastructure. (A–C) Effect of SAM on the morphology of Y79 cells, with a concentration-dependent decrease in nuclear schizophrenic images. (b): Electron microscopic changes in the ultrastructure of retinoblastoma with changes in SAM concentration (12,000×). Control-0mM (a and b): Normal structure of retinoblastoma Y79 cells under transmission electron microscopy. SAM-1mM (Low-dose treatment group: [c and d]): Ultrastructural changes in retinoblastoma Y79 cells. SAM-2mM (high-dose treatment group: [e and f]): Ultrastructural changes in retinoblastoma Y79 cells. Red arrows indicate nuclear grooves, blue arrows denote normal mitochondria, orange arrows represent normal endoplasmic reticulum, yellow arrows point to apoptotic bodies, green arrows indicate swollen, ruptured, vacuolated mitochondria, and purple arrows denote swollen endoplasmic reticulum. Each experiment was repeated for 3 times independently, and the data are presented as mean ± standard deviation. Data in panels were analyzed using one-way ANOVA, nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 (vs. control or low dose administration group). ANOVA, analysis of variance; H&E, Hematoxylin and eosin stain; SAM, S-adenosylmethionine.

Fig 3.

SAM inducing apoptosis and cell cycle arrest of Y79 cells were treated with SAM (1 mM, 2 mM) for 48 h. (A–D) Flow cytometry was used to test the effect of SAM on apoptosis. (B–E) TUNEL assay was carried out to detect the effect of SAM on apoptosis again. (C–F) Flow cytometry was used to test distribution of the apoptotic cells. The percentage of early and late apoptotic cells was assessed and compared to control group. The quantification of the data shows a significant increase of early and late apoptotic cells and a decrease of living cells after 48 h of treatment. N = 3. Each experiment was repeated for 3 times independently, and the data are presented as mean ± standard deviation. Data in panels a–c were statistically analyzed by the using one-way ANOVA. nsP > 0.05, **P < 0.01, ****P < 0.0001 (vs. control or low dose administration group). ANOVA, analysis of variance; SAM, S-adenosylmethionine.
SAM inducing apoptosis and cell cycle arrest of Y79 cells were treated with SAM (1 mM, 2 mM) for 48 h. (A–D) Flow cytometry was used to test the effect of SAM on apoptosis. (B–E) TUNEL assay was carried out to detect the effect of SAM on apoptosis again. (C–F) Flow cytometry was used to test distribution of the apoptotic cells. The percentage of early and late apoptotic cells was assessed and compared to control group. The quantification of the data shows a significant increase of early and late apoptotic cells and a decrease of living cells after 48 h of treatment. N = 3. Each experiment was repeated for 3 times independently, and the data are presented as mean ± standard deviation. Data in panels a–c were statistically analyzed by the using one-way ANOVA. nsP > 0.05, **P < 0.01, ****P < 0.0001 (vs. control or low dose administration group). ANOVA, analysis of variance; SAM, S-adenosylmethionine.

Fig 4.

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. (A): RT-PCR was used to assess the impact of SAM on the mRNA levels of Wnt2, Axin1, β-catenin, c-MYC, cyclin D, c-JUN, and GSK-3β genes. (B and C): RT-PCR was employed to test the effect of SAM on the mRNA level of cell proliferation gene Ki67, metastasis-related genes mmp-2, mmp-9, cell cycle-related genes P21, TP53, vascular endothelial factor VEGF, TGF-β, apoptosis-related genes caspase3, bax, bcl-2. (D and E) Western blot analysis was performed to evaluate the protein levels of Wnt2 and β-catenin. The experiments were conducted with a sample size of N = 3 and repeated independently 3 times. The data are presented as mean ± standard deviation. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). SAM, S-adenosylmethionine; TGF-β, transforming growth factor-β.
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. (A): RT-PCR was used to assess the impact of SAM on the mRNA levels of Wnt2, Axin1, β-catenin, c-MYC, cyclin D, c-JUN, and GSK-3β genes. (B and C): RT-PCR was employed to test the effect of SAM on the mRNA level of cell proliferation gene Ki67, metastasis-related genes mmp-2, mmp-9, cell cycle-related genes P21, TP53, vascular endothelial factor VEGF, TGF-β, apoptosis-related genes caspase3, bax, bcl-2. (D and E) Western blot analysis was performed to evaluate the protein levels of Wnt2 and β-catenin. The experiments were conducted with a sample size of N = 3 and repeated independently 3 times. The data are presented as mean ± standard deviation. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). SAM, S-adenosylmethionine; TGF-β, transforming growth factor-β.

Fig 5.

SAM attenuated the cancer-promoting effects induced by activation of the Wnt2/β-catenin signaling pathway (concentration-dependent attenuation). (A) CCK8 experiments were performed to test the cell proliferation or viability of Y79 cells with the impact of SAM and HLY78 on Y79 cell proliferation. (B) RT-PCR was employed to test the effect of SAM and HLY78 on the expression of mRNA level of Wnt2/β-catenin. (C) The Wnt2/β-catenin and cleaved caspase 3/caspase 3 expressions were determined by western blot. (D) RTPCR was employed to test the expression of mRNA level of related genes. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). CCK8, Cell counting kit-8; SAM, S-adenosylmethionine.
SAM attenuated the cancer-promoting effects induced by activation of the Wnt2/β-catenin signaling pathway (concentration-dependent attenuation). (A) CCK8 experiments were performed to test the cell proliferation or viability of Y79 cells with the impact of SAM and HLY78 on Y79 cell proliferation. (B) RT-PCR was employed to test the effect of SAM and HLY78 on the expression of mRNA level of Wnt2/β-catenin. (C) The Wnt2/β-catenin and cleaved caspase 3/caspase 3 expressions were determined by western blot. (D) RTPCR was employed to test the expression of mRNA level of related genes. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). CCK8, Cell counting kit-8; SAM, S-adenosylmethionine.

Fig 6.

SAM dampened cell proliferation of Y79 cells in vivo. (A) Schematic diagram of the treatment strategy in vivo experiments. SAM treatment led to a significant inhibition of Y79 cell growth in vivo. (B) Y79 cells were subcutaneously injected into the right lower abdomen of BALB/c nude mice to establish a xenograft model, which was then treated according to different groups. Images depict tumors from each group. (C) Changes in tumor volume after each administration. (D) Comparison of body weight changes in nude mice from before treatment initiation to euthanasia. Following SAM treatment, the experimental group exhibited significantly reduced tumor volumes compared to the control group, with an increase in body weight observed. (E and F) The expressions of Ki67 in xenograft tumors were analyzed by immunohistochemistry (original magnification ×200). (G) There were no statistically significant differences in serum indices of liver and kidney function in nude mice. The experiments were repeated independently 3 times with a sample size of N = 4–6, and the data are presented as mean ± standard deviation. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). SAM, S-adenosylmethionine.
SAM dampened cell proliferation of Y79 cells in vivo. (A) Schematic diagram of the treatment strategy in vivo experiments. SAM treatment led to a significant inhibition of Y79 cell growth in vivo. (B) Y79 cells were subcutaneously injected into the right lower abdomen of BALB/c nude mice to establish a xenograft model, which was then treated according to different groups. Images depict tumors from each group. (C) Changes in tumor volume after each administration. (D) Comparison of body weight changes in nude mice from before treatment initiation to euthanasia. Following SAM treatment, the experimental group exhibited significantly reduced tumor volumes compared to the control group, with an increase in body weight observed. (E and F) The expressions of Ki67 in xenograft tumors were analyzed by immunohistochemistry (original magnification ×200). (G) There were no statistically significant differences in serum indices of liver and kidney function in nude mice. The experiments were repeated independently 3 times with a sample size of N = 4–6, and the data are presented as mean ± standard deviation. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (vs. control or low dose administration group). SAM, S-adenosylmethionine.

The primers of RT-PCR

GenesPrimer sequences
GAPDHF: 5′-ACCCACTCCTCCACCTTTGAC-3′
R: 5′-TGTTGCTGTAGCCAAATTCGTT-3′
Ki67F: 5′-ACGCCTGGGTTACTATCAAAAGG-3′
R: 5′-CAGACCCATTTACTTGTGTTGGA-3′
CASPASE 3F: 5′-GAAATTGTGGAATTGATGCGTGA-3′
R: 5′-CTACAACGATCCCCTCTGAAAAA-3′
BaxF: 5′-CCCGAGAGGTCTTTTTCCGAG-3′
R: 5′-CCAGCCCATGATGGTTCTGAT-3′
BCL-2F: 5′-GGTGGGGGTCATGTGTGTGTGG-3′
R: 5′-CGGTTCAGGTACTCAGTCATCC-3′
MMP-2F: 5′-CCCACTGCGGTTTTCTCGAAT-3′
R: 5′-CAAAGGGGGTATCCATCGCCAT-3′
MMP-9F: 5′-TGTACCGCTATGGTTACACTCG-3′
R: 5′-GGCAGGGGACAGTTGCTTCT-3′
VEGFF: 5′-AGGGCAGAATCATCACGAAGT-3′
R: 5′-AGGGTCTCGATTGGATGGCA-3′
TGF-βF: 5′-CTAATGGTGGAAACCCACAACG-3′
R: 5′-TATCGCCAGGAATTGTTGCTG-3′
P21F: 5′-CGATGGAACTTCGACTTTGTCA-3′
R: 5′-GCACAAGGGTACAAGACAGTG-3′
TP53F: 5′-GAGGTTGGGCTCTCTGACTGTACC-3′
R: 5′-TCCGTCCCAGTAGATTACCAC-3
β-CATENINF: 5′-CCTGTTCCCCTGAGGGTATT-3′
R: 5′-CCATCAAATCAGCTTGAGTAGCC-3
Wnt2F: 5′-GCCTTTGTTTATGCCATCTCCT-3′
R: 5′-CTTGGCGCTTCCCATCTTCTT-3′
c-MYCF: 5′-TGCACCCACATCATCTACAG-3′
R: 5′-ACTCGTCATTCCACTCCCAT-3′
cycDF: 5′-AGTGAGCTCAGGAGGAGGTGGTGTAA-3′
R: 5′-AGTAAGCTTGTGAGGGCAGAGGTGTC-3′
c-JUNF: 5′-TCCAAGTGCCGAAAAAGGAAG-3′
R: 5′-CGAGTTCTGAGCTTTCAAGGT-3′
Axin1F: 5′-GACAAGATCGCAGAGGAAGG-3′
R: 5′-ACCCCCACAGTCAAACTCGTC-3′
GSK-3βF: 5′-CCTGGGAACTCCAACAAGGG-3′
R: 5′-GGGGTCGGAAGACCTTAGTC-3′
Language: English
Submitted on: Apr 24, 2024
Accepted on: Aug 5, 2024
Published on: Oct 4, 2024
Published by: Hirszfeld Institute of Immunology and Experimental Therapy
In partnership with: Paradigm Publishing Services
Publication frequency: 1 times per year

© 2024 Mushi Liu, Youchaou Mobet, Hong Shen, published by Hirszfeld Institute of Immunology and Experimental Therapy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.