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Combined effects of 17-DMAG and TNF on cells through a mechanism related to the NF-kappaB pathway.

Qu Z, Dong H, Xu X, Feng W, Yi X - Diagn Pathol (2013)

Bottom Line: This study is to determine the combined effects of 17-DMAG and TNF on malignant cells and the related mechanisms.It was found that the combined treatments resulted in synergistic killing of malignant cells, which was confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258.The immunoblotting results indicated that the synergistic killing due to 17-DMAG and TNF treatments may be related to the decreases in IKKβ levels in the presence of 17-DMAG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong province 266021, China. quzhuling@126.com

ABSTRACT

Objective: The tumor necrosis factor (TNF) and the cellular NF-κB pathway protein IKKβ play important roles in various cellular processes such as cell proliferation, survival, differentiation, and apoptosis. A heat shock protein 90 inhibitor, 17-DMAG, can induce apoptosis of some tumor cells. This study is to determine the combined effects of 17-DMAG and TNF on malignant cells and the related mechanisms.

Methods: We have determined effects of 17-DMAG, an Hsp90 inhibitor, and TNF treatments on the small cell lung cancer cell line (MS-1), the adenocarcinoma cell line (A549), the squamous-cell carcinoma cell line (LK-2), and the normal human bronchial epithelium cell line (NuLi-1) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide assay. To determine if 17-DMAG inhibit the expression of IKKβ in the normal human NuLi-1 cells, and the malignant MS-1, A549, and LK-2 cells, immunoblotting assays and luciferase assays were performed.

Results: It was found that the combined treatments resulted in synergistic killing of malignant cells, which was confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258. The immunoblotting results indicated that the synergistic killing due to 17-DMAG and TNF treatments may be related to the decreases in IKKβ levels in the presence of 17-DMAG.

Conclusions: The results suggest that combination of 17-DMAG and TNF treatments might be useful for treating malignancies upon further study in the further.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041198513886824.

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Related in: MedlinePlus

Cell treatments with DMSO, TNF-α, 17-DMAG, or TNF-α and 17-DMAG together. The normal human bronchial epithelium cell line (NuLi-1) and three lung cancer cell lines (MS-1, A549, and LK-2) were treated with either vehicle control (DMSO), TNF-α (10 ng/ml), 17-DMAG (0.05 μM), or both. Cell counts in each condition were determined by trypan blue exclusion at the time points indicated. (A) NuLi-1 cells; (B) MS-1 cells; (C) A549 cells; (D) LK-2 cells. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) assay immediately before (day 0) and after 1, 2, or 3 days of incubation with the drugs. Values are means ± SD for three experiments. It is considered not significant, when P > 0.05 vs. control (DMSO) cell viability of each treatment. *, it is considered as a significant difference, when P < 0.05 vs. corresponding control.
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Figure 1: Cell treatments with DMSO, TNF-α, 17-DMAG, or TNF-α and 17-DMAG together. The normal human bronchial epithelium cell line (NuLi-1) and three lung cancer cell lines (MS-1, A549, and LK-2) were treated with either vehicle control (DMSO), TNF-α (10 ng/ml), 17-DMAG (0.05 μM), or both. Cell counts in each condition were determined by trypan blue exclusion at the time points indicated. (A) NuLi-1 cells; (B) MS-1 cells; (C) A549 cells; (D) LK-2 cells. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) assay immediately before (day 0) and after 1, 2, or 3 days of incubation with the drugs. Values are means ± SD for three experiments. It is considered not significant, when P > 0.05 vs. control (DMSO) cell viability of each treatment. *, it is considered as a significant difference, when P < 0.05 vs. corresponding control.

Mentions: Results showed that the treatments with the drug vehicle control (DMSO) did not significantly affect cell viability of all of these four types of cells, including the normal human bronchial epithelium cell line (NuLi-1, Figure 1A) and three lung cancer cell lines MS-1 (Figure 1B), A549 (Figure 1C), and LK-2 (Figure 1D). Treatments with TNF-α had slight effects, if any, on cell viability of all of these four types of cells (Figure 1A-D). Treatments with 17-DMAG decreased viabilities of the three lung cancer cell lines MS-1 (Figure 1B), A549 (Figure 1C), and LK-2 (Figure 1D) by approximately 40% at day 2 and up to 60% at day 3, but no obvious decreases for the normal human bronchial epithelium cell line (NuLi-1, Figure 1A). When treated with TNF-α and 17-DMAG together, the viabilities of the three lung cancer cell lines MS-1 (Figure 1B), A549 (Figure 1C), and LK-2 (Figure 1D) were reduced by more than 80% at day 2 and by 90% at day 3. Combined treatments with TNF-α and 17-DMAG had not significantly decreased the viability of the normal cells (NuLi-1, Figure 1A), suggesting that such dosages of TNF-α and 17-DMAG are not toxic to normal cells. The above results suggest that TNF-α enhances the toxic effects on tumor cells of 17-DMAG.


Combined effects of 17-DMAG and TNF on cells through a mechanism related to the NF-kappaB pathway.

Qu Z, Dong H, Xu X, Feng W, Yi X - Diagn Pathol (2013)

Cell treatments with DMSO, TNF-α, 17-DMAG, or TNF-α and 17-DMAG together. The normal human bronchial epithelium cell line (NuLi-1) and three lung cancer cell lines (MS-1, A549, and LK-2) were treated with either vehicle control (DMSO), TNF-α (10 ng/ml), 17-DMAG (0.05 μM), or both. Cell counts in each condition were determined by trypan blue exclusion at the time points indicated. (A) NuLi-1 cells; (B) MS-1 cells; (C) A549 cells; (D) LK-2 cells. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) assay immediately before (day 0) and after 1, 2, or 3 days of incubation with the drugs. Values are means ± SD for three experiments. It is considered not significant, when P > 0.05 vs. control (DMSO) cell viability of each treatment. *, it is considered as a significant difference, when P < 0.05 vs. corresponding control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3716826&req=5

Figure 1: Cell treatments with DMSO, TNF-α, 17-DMAG, or TNF-α and 17-DMAG together. The normal human bronchial epithelium cell line (NuLi-1) and three lung cancer cell lines (MS-1, A549, and LK-2) were treated with either vehicle control (DMSO), TNF-α (10 ng/ml), 17-DMAG (0.05 μM), or both. Cell counts in each condition were determined by trypan blue exclusion at the time points indicated. (A) NuLi-1 cells; (B) MS-1 cells; (C) A549 cells; (D) LK-2 cells. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) assay immediately before (day 0) and after 1, 2, or 3 days of incubation with the drugs. Values are means ± SD for three experiments. It is considered not significant, when P > 0.05 vs. control (DMSO) cell viability of each treatment. *, it is considered as a significant difference, when P < 0.05 vs. corresponding control.
Mentions: Results showed that the treatments with the drug vehicle control (DMSO) did not significantly affect cell viability of all of these four types of cells, including the normal human bronchial epithelium cell line (NuLi-1, Figure 1A) and three lung cancer cell lines MS-1 (Figure 1B), A549 (Figure 1C), and LK-2 (Figure 1D). Treatments with TNF-α had slight effects, if any, on cell viability of all of these four types of cells (Figure 1A-D). Treatments with 17-DMAG decreased viabilities of the three lung cancer cell lines MS-1 (Figure 1B), A549 (Figure 1C), and LK-2 (Figure 1D) by approximately 40% at day 2 and up to 60% at day 3, but no obvious decreases for the normal human bronchial epithelium cell line (NuLi-1, Figure 1A). When treated with TNF-α and 17-DMAG together, the viabilities of the three lung cancer cell lines MS-1 (Figure 1B), A549 (Figure 1C), and LK-2 (Figure 1D) were reduced by more than 80% at day 2 and by 90% at day 3. Combined treatments with TNF-α and 17-DMAG had not significantly decreased the viability of the normal cells (NuLi-1, Figure 1A), suggesting that such dosages of TNF-α and 17-DMAG are not toxic to normal cells. The above results suggest that TNF-α enhances the toxic effects on tumor cells of 17-DMAG.

Bottom Line: This study is to determine the combined effects of 17-DMAG and TNF on malignant cells and the related mechanisms.It was found that the combined treatments resulted in synergistic killing of malignant cells, which was confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258.The immunoblotting results indicated that the synergistic killing due to 17-DMAG and TNF treatments may be related to the decreases in IKKβ levels in the presence of 17-DMAG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong province 266021, China. quzhuling@126.com

ABSTRACT

Objective: The tumor necrosis factor (TNF) and the cellular NF-κB pathway protein IKKβ play important roles in various cellular processes such as cell proliferation, survival, differentiation, and apoptosis. A heat shock protein 90 inhibitor, 17-DMAG, can induce apoptosis of some tumor cells. This study is to determine the combined effects of 17-DMAG and TNF on malignant cells and the related mechanisms.

Methods: We have determined effects of 17-DMAG, an Hsp90 inhibitor, and TNF treatments on the small cell lung cancer cell line (MS-1), the adenocarcinoma cell line (A549), the squamous-cell carcinoma cell line (LK-2), and the normal human bronchial epithelium cell line (NuLi-1) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide assay. To determine if 17-DMAG inhibit the expression of IKKβ in the normal human NuLi-1 cells, and the malignant MS-1, A549, and LK-2 cells, immunoblotting assays and luciferase assays were performed.

Results: It was found that the combined treatments resulted in synergistic killing of malignant cells, which was confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258. The immunoblotting results indicated that the synergistic killing due to 17-DMAG and TNF treatments may be related to the decreases in IKKβ levels in the presence of 17-DMAG.

Conclusions: The results suggest that combination of 17-DMAG and TNF treatments might be useful for treating malignancies upon further study in the further.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041198513886824.

Show MeSH
Related in: MedlinePlus