TLDR: Methylsulfonylmethane kills cancer stem cells by lowering intracellular iron.
Side note: MSM is non toxic to normal cells, tested up to 8g/kg/day in mice(HED: 650mg/kg/day)
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“We used increasing concentrations of MSM in NCCIT (Supplementary Figure S1A) and NTERA-2 (Supplementary Figure S1B) and found a concentration-dependent inhibition in cell viability by MSM.”
“Then, we checked whether MSM could inhibit CSC markers in embryonic CSCs. Analysis of CSC markers SOX2, OCT4, and NANOG in NCCIT, and NTERA-2 cells upon MSM treatment, showed downregulation in the expression patterns of CSC markers (Figure 1A). We confirmed the inhibition of CSC markers by MSM at the mRNA level using real-time qPCR analysis (Figure 1B). These results suggested the ability of MSM to inhibit CSC proliferation”
“Then, we analyzed the expression patterns of Wnt/β-catenin signaling in embryonic CSC in the presence of MSM. The obtained results indicated an inhibition in the expression levels of Wnt5a, Wnt8A, GSK-3β, β-catenin, and TCF proteins by MSM treatment in NCCIT and NTERA-2 cells (Figure 1C). We then confirmed the inhibition of Wnt/β-catenin signaling in mRNA level by MSM treatment in embryonic CSC (Figure 1D). These results indicated the capability of MSM against CSCs.”
“we assumed that the anticancer activity of MSM might begin by inducing ROS formation. To analyze this, first, we checked the expression pattern of iNOS, which can generate ROS. Our results showed that increasing concentrations of MSM induced the expression levels of iNOS protein in NCCIT and NTERA-2 cells”
“Furthermore, we found that MSM successfully generated cellular ROS (Figure 2C) and mitochondrial ROS (Figure 2D), suggesting that the anticancer activity of MSM is due to ROS generation.”
“We then checked the ability of MSM to induce DNA damage response (DDR) in embryonic CSC. To analyze this, we used a comet assay to determine DNA double-strand breaks and our results showed that MSM induced the DNA double-strand break in NCCIT and NTERA-2 cells (Supplementary Figure S2A). Furthermore, we observed a significant increase in comet length and positive cells in MSM-treated cells compared with nontreated control cells (Supplementary Figure S2B). These results hinted at DDR induction by MSM in embryonic CSC. To confirm this, we analyzed the expression patterns of proteins responsible for DDR and found an elevation in the expression of phosphorylated ATM, ATR, CHK1, CHK2, and BRCA1 by MSM treatment in NCCIT and NTERA-2 cells (Supplementary Figure S2C). These results suggested that ATM or ATR acts as a key regulator in DDR induction by MSM.”
“On the basis of previous results, we showed that MSM could generate ROS and induce DDR in embryonic CSC.”
“First, the analysis of cell cycle in embryonic CSC with or without 200 mM MSM and the obtained flow cytometry results showed an arrest in the G0/G1 phase of the cell cycle by MSM (Supplementary Figure S3A). This result indicated that induced DDR results in prolonged cell cycle arrest”
“We found an elevation in the expression levels of tumor suppressor genes, CDKN1A and CDKN1B, and a decrease in the expression levels of CCND1, CCNE1, and CDK4 genes (Supplementary Figure S3B). Protein levels confirmed these results by analyzing the expression pattern of cyclin D1, cyclin E, CDK4, p21, and p27 by Western blotting (Supplementary Figure S3C). These results showed cell cycle arrest by MSM in embryonic CSC and suggested a possible induction of apoptosis by MSM.”
“As we found the induction of apoptosis by MSM, we then investigated the apoptosis pathway by checking the key apoptosis regulators, BCL2-associated X (BAX), B-cell lymphoma 2 (BCL-2), and cytochrome C proteins. However, the obtained result was opposite to our expectations as expression levels of BAX and cytochrome C downregulated with increased or unchanged BCL-2 expression in embryonic CSC (Supplementary Figure S4). This forced us to analyze the genes that take part in the extrinsic pathway of apoptosis, and we found an elevation in the expression levels of TRAIL protein along with its receptors, DR4 and DR5, and its downstream mediator, TRADD, and then the upregulation of caspase-8 and -3 precursor proteins (Figure 3B). To confirm the activation of the TRAIL-mediated extrinsic apoptosis pathway by MSM, we analyzed mRNA levels of genes present in the extrinsic apoptosis pathway and found an elevation in the expression levels of all genes (Figure 3C). These results suggested that MSM induced TRAIL-mediated extrinsic apoptosis. We also confirmed the induction of TRAIL by MSM by Human TRAIL assay and observed a significant increase in TRAIL expression (Figure 3D). We then confirmed the extrinsic apoptosis induction by analyzing caspase 3/7 expression and obtained the result indicating a significant elevation of caspase 3/7 by 200-mM MSM in NCCIT and NTERA-2 cells (Figure 3E). Hence, it is confirmed that MSM induced TRAIL-dependent extrinsic apoptosis in embryonic CSC.”
“We found that MSM can induce apoptosis in embryonic CSCs. Here, we analyzed the mechanistic aspect behind these activities and assumed that iron metabolism might have played a crucial role. First, we estimated iron concentration in cells and media with or without MSM treatment using an Iron Assay Kit (Figure 4A). The results for NCCIT and NTERA-2 cells showed an increase in total iron concentration in media by MSM treatment, whereas, in MSM-treated cells, the concentration of total iron decreased significantly in both cells. This showed the enhancement of iron release from cells to the media.”
“Then, we confirmed this by estimating ferrous ion (Fe2+) concentration in NCCIT and NTERA-2 cells after treatment with MSM by flow cytometry, and results suggested a significant decrease in Fe2+ ion concentration (Figure 4B). These results indicated an upregulation in iron transport by converting Fe2+ to ferric ion (Fe3+), which highlighted the conversion of total iron for iron metabolism”
“To confirm this, we analyzed the expression levels of proteins responsible for iron transport, and the results showed downregulation in the expression levels of transferrin receptor (TFR1) and ferroportin (FPN1) that helps iron intake and outtake (Figure 4C). We also found that MSM suppressed the expression levels of divalent metal transporter 1 (DMT1) and six-transmembrane epithelial antigen of prostate 3 (STEAP3), helping iron conversion. These results suggested the role of iron metabolism in the anticancer activity of MSM against embryonic CSCs.”
“We found downregulated expression of TRAIL and phosphorylated p38 expression upon SB 203580 treatment with no cytotoxicity effect, and MSM successfully elevated these expressions, suggesting the ability of MSM to induce TRAIL expression by p38 upregulation”
“We also confirmed the role of ERK in TRAIL expression using a specific inhibitor of ERK phosphorylation (PD 98,059) with or without MSM in embryonic CSC, and the results showed an increase in the expression of TRAIL upon PD 98,059 treatment with no cytotoxicity effect; MSM addition further increased TRAIL expression in both cells (Figure 5C). These results also indicated that MSM induced TRAIL expression by downregulating ERK expression.”
“Then, we analyzed the role of iron metabolism in TRAIL induction and the p38/ERK/p53 signaling pathway. For this, we used iron sulfate (FeSO4) in NCCIT and NTERA-2 cells with or without MSM treatment. The results showed suppression of TRAIL, p53, and phospho-p38 expression levels and an elevation in the expression levels of phospho-ERK by FeSO4 treatment with no cytotoxicity effect (Figure 5D). MSM activity successfully reversed these expressions, which suggested the role of iron metabolism in the TRAIL expression by MSM treatment in embryonic CSCs.”
“The obtained result from mRNA analysis showed an upregulation in the expression of miR-130a and downregulation in the expression of miR-221 and miR-222 by MSM treatment in NCCIT and NTERA-2 cells”
“The results showed suppression in the expression of miR-130a by the anti-miR treatment, which was then elevated by MSM treatment, whereas miR-221 and miR-22 expression was further downregulated followed by MSM treatment. These results showed the ability of MSM to regulate these microRNAs”
“we checked the role of iron metabolism in the expression of these microRNAs using FeSO4 treatment with or without MSM. The result showed a decrease in the expression of miR-130a and an increase in the expression of miR-221 and miR-222 by FeSO4 treatment, which were successfully reversed by MSM treatment in both cells”
“Altogether, the molecular mechanism behind the apoptosis induction of MSM begins with the inhibition of iron metabolism, which then induces ROS formation and thereby regulates p38/ERK/p53 signaling, which activates the TRAIL-mediated extrinsic pathway of apoptosis”
“Therefore, considering MSM to treat embryonic CSCs may provide the best knowledge of its ability to target CSCs without affecting the growth of normal cells.”
Side note: MSM is non toxic to normal cells, tested up to 8g/kg/day in mice(HED: 650mg/kg/day)
Iron Metabolism as a Potential Mechanism for Inducing TRAIL-Mediated Extrinsic Apoptosis Using Methylsulfonylmethane in Embryonic Cancer Stem Cells
Embryonic cancer stem cells (CSCs) can differentiate into any cancer type. Targeting CSC using natural compounds is a good approach as it suppresses cancer recurrence with fewer adverse effects, and methylsulfonylmethane (MSM) is a sulfur-containing compound with well-known anticancer...
www.mdpi.com
“We used increasing concentrations of MSM in NCCIT (Supplementary Figure S1A) and NTERA-2 (Supplementary Figure S1B) and found a concentration-dependent inhibition in cell viability by MSM.”
“Then, we checked whether MSM could inhibit CSC markers in embryonic CSCs. Analysis of CSC markers SOX2, OCT4, and NANOG in NCCIT, and NTERA-2 cells upon MSM treatment, showed downregulation in the expression patterns of CSC markers (Figure 1A). We confirmed the inhibition of CSC markers by MSM at the mRNA level using real-time qPCR analysis (Figure 1B). These results suggested the ability of MSM to inhibit CSC proliferation”
“Then, we analyzed the expression patterns of Wnt/β-catenin signaling in embryonic CSC in the presence of MSM. The obtained results indicated an inhibition in the expression levels of Wnt5a, Wnt8A, GSK-3β, β-catenin, and TCF proteins by MSM treatment in NCCIT and NTERA-2 cells (Figure 1C). We then confirmed the inhibition of Wnt/β-catenin signaling in mRNA level by MSM treatment in embryonic CSC (Figure 1D). These results indicated the capability of MSM against CSCs.”
“we assumed that the anticancer activity of MSM might begin by inducing ROS formation. To analyze this, first, we checked the expression pattern of iNOS, which can generate ROS. Our results showed that increasing concentrations of MSM induced the expression levels of iNOS protein in NCCIT and NTERA-2 cells”
“Furthermore, we found that MSM successfully generated cellular ROS (Figure 2C) and mitochondrial ROS (Figure 2D), suggesting that the anticancer activity of MSM is due to ROS generation.”
“We then checked the ability of MSM to induce DNA damage response (DDR) in embryonic CSC. To analyze this, we used a comet assay to determine DNA double-strand breaks and our results showed that MSM induced the DNA double-strand break in NCCIT and NTERA-2 cells (Supplementary Figure S2A). Furthermore, we observed a significant increase in comet length and positive cells in MSM-treated cells compared with nontreated control cells (Supplementary Figure S2B). These results hinted at DDR induction by MSM in embryonic CSC. To confirm this, we analyzed the expression patterns of proteins responsible for DDR and found an elevation in the expression of phosphorylated ATM, ATR, CHK1, CHK2, and BRCA1 by MSM treatment in NCCIT and NTERA-2 cells (Supplementary Figure S2C). These results suggested that ATM or ATR acts as a key regulator in DDR induction by MSM.”
“On the basis of previous results, we showed that MSM could generate ROS and induce DDR in embryonic CSC.”
“First, the analysis of cell cycle in embryonic CSC with or without 200 mM MSM and the obtained flow cytometry results showed an arrest in the G0/G1 phase of the cell cycle by MSM (Supplementary Figure S3A). This result indicated that induced DDR results in prolonged cell cycle arrest”
“We found an elevation in the expression levels of tumor suppressor genes, CDKN1A and CDKN1B, and a decrease in the expression levels of CCND1, CCNE1, and CDK4 genes (Supplementary Figure S3B). Protein levels confirmed these results by analyzing the expression pattern of cyclin D1, cyclin E, CDK4, p21, and p27 by Western blotting (Supplementary Figure S3C). These results showed cell cycle arrest by MSM in embryonic CSC and suggested a possible induction of apoptosis by MSM.”
“As we found the induction of apoptosis by MSM, we then investigated the apoptosis pathway by checking the key apoptosis regulators, BCL2-associated X (BAX), B-cell lymphoma 2 (BCL-2), and cytochrome C proteins. However, the obtained result was opposite to our expectations as expression levels of BAX and cytochrome C downregulated with increased or unchanged BCL-2 expression in embryonic CSC (Supplementary Figure S4). This forced us to analyze the genes that take part in the extrinsic pathway of apoptosis, and we found an elevation in the expression levels of TRAIL protein along with its receptors, DR4 and DR5, and its downstream mediator, TRADD, and then the upregulation of caspase-8 and -3 precursor proteins (Figure 3B). To confirm the activation of the TRAIL-mediated extrinsic apoptosis pathway by MSM, we analyzed mRNA levels of genes present in the extrinsic apoptosis pathway and found an elevation in the expression levels of all genes (Figure 3C). These results suggested that MSM induced TRAIL-mediated extrinsic apoptosis. We also confirmed the induction of TRAIL by MSM by Human TRAIL assay and observed a significant increase in TRAIL expression (Figure 3D). We then confirmed the extrinsic apoptosis induction by analyzing caspase 3/7 expression and obtained the result indicating a significant elevation of caspase 3/7 by 200-mM MSM in NCCIT and NTERA-2 cells (Figure 3E). Hence, it is confirmed that MSM induced TRAIL-dependent extrinsic apoptosis in embryonic CSC.”
“We found that MSM can induce apoptosis in embryonic CSCs. Here, we analyzed the mechanistic aspect behind these activities and assumed that iron metabolism might have played a crucial role. First, we estimated iron concentration in cells and media with or without MSM treatment using an Iron Assay Kit (Figure 4A). The results for NCCIT and NTERA-2 cells showed an increase in total iron concentration in media by MSM treatment, whereas, in MSM-treated cells, the concentration of total iron decreased significantly in both cells. This showed the enhancement of iron release from cells to the media.”
“Then, we confirmed this by estimating ferrous ion (Fe2+) concentration in NCCIT and NTERA-2 cells after treatment with MSM by flow cytometry, and results suggested a significant decrease in Fe2+ ion concentration (Figure 4B). These results indicated an upregulation in iron transport by converting Fe2+ to ferric ion (Fe3+), which highlighted the conversion of total iron for iron metabolism”
“To confirm this, we analyzed the expression levels of proteins responsible for iron transport, and the results showed downregulation in the expression levels of transferrin receptor (TFR1) and ferroportin (FPN1) that helps iron intake and outtake (Figure 4C). We also found that MSM suppressed the expression levels of divalent metal transporter 1 (DMT1) and six-transmembrane epithelial antigen of prostate 3 (STEAP3), helping iron conversion. These results suggested the role of iron metabolism in the anticancer activity of MSM against embryonic CSCs.”
“We found downregulated expression of TRAIL and phosphorylated p38 expression upon SB 203580 treatment with no cytotoxicity effect, and MSM successfully elevated these expressions, suggesting the ability of MSM to induce TRAIL expression by p38 upregulation”
“We also confirmed the role of ERK in TRAIL expression using a specific inhibitor of ERK phosphorylation (PD 98,059) with or without MSM in embryonic CSC, and the results showed an increase in the expression of TRAIL upon PD 98,059 treatment with no cytotoxicity effect; MSM addition further increased TRAIL expression in both cells (Figure 5C). These results also indicated that MSM induced TRAIL expression by downregulating ERK expression.”
“Then, we analyzed the role of iron metabolism in TRAIL induction and the p38/ERK/p53 signaling pathway. For this, we used iron sulfate (FeSO4) in NCCIT and NTERA-2 cells with or without MSM treatment. The results showed suppression of TRAIL, p53, and phospho-p38 expression levels and an elevation in the expression levels of phospho-ERK by FeSO4 treatment with no cytotoxicity effect (Figure 5D). MSM activity successfully reversed these expressions, which suggested the role of iron metabolism in the TRAIL expression by MSM treatment in embryonic CSCs.”
“The obtained result from mRNA analysis showed an upregulation in the expression of miR-130a and downregulation in the expression of miR-221 and miR-222 by MSM treatment in NCCIT and NTERA-2 cells”
“The results showed suppression in the expression of miR-130a by the anti-miR treatment, which was then elevated by MSM treatment, whereas miR-221 and miR-22 expression was further downregulated followed by MSM treatment. These results showed the ability of MSM to regulate these microRNAs”
“we checked the role of iron metabolism in the expression of these microRNAs using FeSO4 treatment with or without MSM. The result showed a decrease in the expression of miR-130a and an increase in the expression of miR-221 and miR-222 by FeSO4 treatment, which were successfully reversed by MSM treatment in both cells”
“Altogether, the molecular mechanism behind the apoptosis induction of MSM begins with the inhibition of iron metabolism, which then induces ROS formation and thereby regulates p38/ERK/p53 signaling, which activates the TRAIL-mediated extrinsic pathway of apoptosis”
“Therefore, considering MSM to treat embryonic CSCs may provide the best knowledge of its ability to target CSCs without affecting the growth of normal cells.”
