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Inhibition of malignant thyroid carcinoma cell proliferation by Ras and galectin-3 inhibitors

View Article: PubMed Central - PubMed

ABSTRACT

Anaplastic Thyroid carcinoma is an extremely aggressive solid tumor that resists most treatments and is almost always fatal. Galectin-3 (Gal-3) is an important marker for thyroid carcinomas and a scaffold of the K-Ras protein. S-trans, transfarnesylthiosalicylic acid (FTS; Salirasib) is a Ras inhibitor that inhibits the active forms of Ras proteins. Modified citrus pectin (MCP) is a water-soluble citrus-fruit-derived polysaccharide fiber that specifically inhibits Gal-3. The aim of this study was to develop a novel drug combination designed to treat aggressive anaplastic thyroid carcinoma. Combined treatment with FTS and MCP inhibited anaplastic thyroid cells proliferation in vitro by inducing cell cycle arrest and increasing apoptosis rate. Immunoblot analysis revealed a significant decrease in Pan-Ras, K-Ras, Ras-GTP, p-ERK, p53, and Gal-3 expression levels and significant increase in p21 expression levels. In nude mice, treatment with FTS and MCP inhibited tumor growth. Levels of Gal-3, K-Ras-GTP, and p-ERK were significantly decreased. To conclude, our results suggest K-Ras and Gal-3 as potential targets in anaplastic thyroid tumors and herald a novel treatment for highly aggressive anaplastic thyroid carcinoma.

No MeSH data available.


Related in: MedlinePlus

Combined treatment of FTS and MCP regulate Ras and its downstream signaling molecules. Combined treatment of FTS and MCP decreases the expression levels of, Galectin-3, Pan-Ras, K-Ras, Pan-Ras-GTP, p-ERK and p53, and increased the expression levels of p21. ARO cells were plated and treated with 75 μM FTS and 0.35% MCP, as described in Methods and Materials. After 48/72 h, cells were lysed and subjected to immunoblotting. (a) Representative Immunoblots of Gal-3, Pan-Ras, and K-Ras normalized to β-tubulin, and Pan-Ras-GTP visualized by ECL. (b) Apparent levels of Galectin-3, Pan-Ras, K-Ras and Pan-Ras-GTP determined by densitometry (a.u.) of the immunoblots. Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (c) Representative Immunoblots of total ERK and p-ERK normalized to actin, visualized by ECL (d) Apparent levels of total ERK and p-ERK determined by densitometry (a.u.) of the immunoblots. P-ERK levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). (e) Immunoblots for p21 and p53 normalized to actin. (f) Apparent levels of p21 and p53 determined by densitometry (a.u.). p21 levels were significantly increased and p53 levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Results are presented as means±S.E.M.; one-way ANOVA analysis revealed significant differences between the groups; post hoc analysis was performed by the HSD test *P<0.05, **P<0.01, ***P<0.001.
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fig2: Combined treatment of FTS and MCP regulate Ras and its downstream signaling molecules. Combined treatment of FTS and MCP decreases the expression levels of, Galectin-3, Pan-Ras, K-Ras, Pan-Ras-GTP, p-ERK and p53, and increased the expression levels of p21. ARO cells were plated and treated with 75 μM FTS and 0.35% MCP, as described in Methods and Materials. After 48/72 h, cells were lysed and subjected to immunoblotting. (a) Representative Immunoblots of Gal-3, Pan-Ras, and K-Ras normalized to β-tubulin, and Pan-Ras-GTP visualized by ECL. (b) Apparent levels of Galectin-3, Pan-Ras, K-Ras and Pan-Ras-GTP determined by densitometry (a.u.) of the immunoblots. Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (c) Representative Immunoblots of total ERK and p-ERK normalized to actin, visualized by ECL (d) Apparent levels of total ERK and p-ERK determined by densitometry (a.u.) of the immunoblots. P-ERK levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). (e) Immunoblots for p21 and p53 normalized to actin. (f) Apparent levels of p21 and p53 determined by densitometry (a.u.). p21 levels were significantly increased and p53 levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Results are presented as means±S.E.M.; one-way ANOVA analysis revealed significant differences between the groups; post hoc analysis was performed by the HSD test *P<0.05, **P<0.01, ***P<0.001.

Mentions: To study the effect of combined treatment with FTS and MCP on protein expression, we treated ARO cells with FTS and MCP for 48 h and assessed protein expression by immunoblot analysis. Ras-GTP levels were assessed by GTPase pull-down assays, as described in Materials and Methods. As shown in Figures 2a and b, treatment with FTS+MCP significantly decreased Ras-GTP and total Ras levels by 37 and 46%, respectively (P<0.01, one-way ANOVA; P<0.01, Tukey's honest significant difference (HSD) test), and total K-Ras levels by 62% (P<0.001, one-way ANOVA; P<0.001, Tukey's HSD test) relative to untreated cells. To further assess the influence of combined treatment on Ras signaling pathways, we examined phospho-ERK (p-ERK), an important downstream protein in Raf/MEK/ERK pathway expression levels (Figure 2c). We found that although FTS+MCP treatment affected total ERK slightly (Figure 2d; P>0.05, one-way ANOVA), it significantly reduced p-ERK expression levels by 60% relative to untreated cells (Figure 2d; P<0.05, One-way ANOVA; P<0.05, Tukey's HSD test). As p-Akt is not detected in ARO cells, it was not used as readout for Ras signaling.23


Inhibition of malignant thyroid carcinoma cell proliferation by Ras and galectin-3 inhibitors
Combined treatment of FTS and MCP regulate Ras and its downstream signaling molecules. Combined treatment of FTS and MCP decreases the expression levels of, Galectin-3, Pan-Ras, K-Ras, Pan-Ras-GTP, p-ERK and p53, and increased the expression levels of p21. ARO cells were plated and treated with 75 μM FTS and 0.35% MCP, as described in Methods and Materials. After 48/72 h, cells were lysed and subjected to immunoblotting. (a) Representative Immunoblots of Gal-3, Pan-Ras, and K-Ras normalized to β-tubulin, and Pan-Ras-GTP visualized by ECL. (b) Apparent levels of Galectin-3, Pan-Ras, K-Ras and Pan-Ras-GTP determined by densitometry (a.u.) of the immunoblots. Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (c) Representative Immunoblots of total ERK and p-ERK normalized to actin, visualized by ECL (d) Apparent levels of total ERK and p-ERK determined by densitometry (a.u.) of the immunoblots. P-ERK levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). (e) Immunoblots for p21 and p53 normalized to actin. (f) Apparent levels of p21 and p53 determined by densitometry (a.u.). p21 levels were significantly increased and p53 levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Results are presented as means±S.E.M.; one-way ANOVA analysis revealed significant differences between the groups; post hoc analysis was performed by the HSD test *P<0.05, **P<0.01, ***P<0.001.
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fig2: Combined treatment of FTS and MCP regulate Ras and its downstream signaling molecules. Combined treatment of FTS and MCP decreases the expression levels of, Galectin-3, Pan-Ras, K-Ras, Pan-Ras-GTP, p-ERK and p53, and increased the expression levels of p21. ARO cells were plated and treated with 75 μM FTS and 0.35% MCP, as described in Methods and Materials. After 48/72 h, cells were lysed and subjected to immunoblotting. (a) Representative Immunoblots of Gal-3, Pan-Ras, and K-Ras normalized to β-tubulin, and Pan-Ras-GTP visualized by ECL. (b) Apparent levels of Galectin-3, Pan-Ras, K-Ras and Pan-Ras-GTP determined by densitometry (a.u.) of the immunoblots. Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Gal-3, Pan-Ras, K-Ras and Pan-Ras-GTP levels were significantly decreased in FTS+MCP-treated cells (c) Representative Immunoblots of total ERK and p-ERK normalized to actin, visualized by ECL (d) Apparent levels of total ERK and p-ERK determined by densitometry (a.u.) of the immunoblots. P-ERK levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). (e) Immunoblots for p21 and p53 normalized to actin. (f) Apparent levels of p21 and p53 determined by densitometry (a.u.). p21 levels were significantly increased and p53 levels were significantly decreased in FTS+MCP-treated cells (white bar) compared with untreated cells (black bar), FTS-treated cells (gray bar) and MCP-treated cells (upward diagonal cells). Results are presented as means±S.E.M.; one-way ANOVA analysis revealed significant differences between the groups; post hoc analysis was performed by the HSD test *P<0.05, **P<0.01, ***P<0.001.
Mentions: To study the effect of combined treatment with FTS and MCP on protein expression, we treated ARO cells with FTS and MCP for 48 h and assessed protein expression by immunoblot analysis. Ras-GTP levels were assessed by GTPase pull-down assays, as described in Materials and Methods. As shown in Figures 2a and b, treatment with FTS+MCP significantly decreased Ras-GTP and total Ras levels by 37 and 46%, respectively (P<0.01, one-way ANOVA; P<0.01, Tukey's honest significant difference (HSD) test), and total K-Ras levels by 62% (P<0.001, one-way ANOVA; P<0.001, Tukey's HSD test) relative to untreated cells. To further assess the influence of combined treatment on Ras signaling pathways, we examined phospho-ERK (p-ERK), an important downstream protein in Raf/MEK/ERK pathway expression levels (Figure 2c). We found that although FTS+MCP treatment affected total ERK slightly (Figure 2d; P>0.05, one-way ANOVA), it significantly reduced p-ERK expression levels by 60% relative to untreated cells (Figure 2d; P<0.05, One-way ANOVA; P<0.05, Tukey's HSD test). As p-Akt is not detected in ARO cells, it was not used as readout for Ras signaling.23

View Article: PubMed Central - PubMed

ABSTRACT

Anaplastic Thyroid carcinoma is an extremely aggressive solid tumor that resists most treatments and is almost always fatal. Galectin-3 (Gal-3) is an important marker for thyroid carcinomas and a scaffold of the K-Ras protein. S-trans, transfarnesylthiosalicylic acid (FTS; Salirasib) is a Ras inhibitor that inhibits the active forms of Ras proteins. Modified citrus pectin (MCP) is a water-soluble citrus-fruit-derived polysaccharide fiber that specifically inhibits Gal-3. The aim of this study was to develop a novel drug combination designed to treat aggressive anaplastic thyroid carcinoma. Combined treatment with FTS and MCP inhibited anaplastic thyroid cells proliferation in vitro by inducing cell cycle arrest and increasing apoptosis rate. Immunoblot analysis revealed a significant decrease in Pan-Ras, K-Ras, Ras-GTP, p-ERK, p53, and Gal-3 expression levels and significant increase in p21 expression levels. In nude mice, treatment with FTS and MCP inhibited tumor growth. Levels of Gal-3, K-Ras-GTP, and p-ERK were significantly decreased. To conclude, our results suggest K-Ras and Gal-3 as potential targets in anaplastic thyroid tumors and herald a novel treatment for highly aggressive anaplastic thyroid carcinoma.

No MeSH data available.


Related in: MedlinePlus