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Specificity protein 1 is a novel target of 2,4-bis (p-hydroxyphenyl)-2-butenal for the suppression of human oral squamous cell carcinoma cell growth.

Chae JI, Lee R, Cho J, Hong J, Shim JH - J. Biomed. Sci. (2014)

Bottom Line: The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing.HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies.Sp1 was significantly inhibited by HPB242 in a dose-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacy, College of Pharmacy, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam 534-729, Republic of Korea. s1004jh@gmail.com.

ABSTRACT

Background: The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects though 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. The purpose of this study was to investigate the anti-proliferative effects of 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) on two oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, through regulation of specificity protein 1 (Sp1).

Results: HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies. Sp1 was significantly inhibited by HPB242 in a dose-dependent manner. Furthermore, cell cycle regulating proteins and anti-apoptotic proteins, which are known as Sp1 target genes, were altered at the molecular levels. The key important regulators in the cell cycle such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, myeloid leukemia sequence 1 (Mcl-1) and survivin were significantly decreased by HPB242 or suppressed Sp1 levels, however pro-apoptotic proteins caspase3 and PARP were cleaved in HN22 and HSC4.

Conclusions: HPB242 may be useful as a chemotherapeutic agent for OSCC for the purpose of treatment and prevention of oral cancer and for the improvement of clinical outcomes.

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Related in: MedlinePlus

The effect of HPB242 on specificity protein 1 (Sp1) protein expression in HN22 and HSC4 cells. HN22 and HSC4 cells were incubated with different concentrations of HPB242 for 48 hours. The cells were harvested and prepared for western blots as described under Methods. Protein expression levels of Sp1 were detected using a specific antibody against Sp1, and its levels were quantified after actin normalization. (A) The HPB242-treated cells were compared with untreated cells, and data are shown as the means ± SD of three independent experiments. The asterisk indicates a significant difference compared with the negative control (untreated cells) (*p < 0.05). (B) Time-dependent effects of HPB242 on Sp1 and Cleaved-caspase3 expression were performed in HN22 and HSC4 cells for 48 hours with 6 hours intervals. (C) The effect of HPB242 (0–20 μg/ml) for 48 hours on Sp1 mRNA expression was determined by RT-PCR. The graphs indicate the ratio of Sp1 to β-actin expression. (D) The effect of HPB242 on Sp1 protein turnover in HN22 and HSC4 cells. The protein lysates were obtained from cells pretreated with protein synthesis inhibitor such as cycloheximide (CHX) for 2 hour and then exposed to HPB242 for 48 hours. The protein expression of Sp1 was analyzed by western blot analysis. (E) Immunocytochemistry analysis was performed in HPB242 treated HN22 and HSC4 cells. HN22 and HSC4 cells were treated with different concentrations of HPB242 for 48 hours, and cells were immunostained with Sp1 specific antibody, Cleaved-caspase3 specific antibody, and then signals were detected with Jackson 488- and 647-conjugated anti-mouse secondary antibody. DAPI was used for nucleus staining. 254×190 mm (96 × 96 DPI).
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Figure 3: The effect of HPB242 on specificity protein 1 (Sp1) protein expression in HN22 and HSC4 cells. HN22 and HSC4 cells were incubated with different concentrations of HPB242 for 48 hours. The cells were harvested and prepared for western blots as described under Methods. Protein expression levels of Sp1 were detected using a specific antibody against Sp1, and its levels were quantified after actin normalization. (A) The HPB242-treated cells were compared with untreated cells, and data are shown as the means ± SD of three independent experiments. The asterisk indicates a significant difference compared with the negative control (untreated cells) (*p < 0.05). (B) Time-dependent effects of HPB242 on Sp1 and Cleaved-caspase3 expression were performed in HN22 and HSC4 cells for 48 hours with 6 hours intervals. (C) The effect of HPB242 (0–20 μg/ml) for 48 hours on Sp1 mRNA expression was determined by RT-PCR. The graphs indicate the ratio of Sp1 to β-actin expression. (D) The effect of HPB242 on Sp1 protein turnover in HN22 and HSC4 cells. The protein lysates were obtained from cells pretreated with protein synthesis inhibitor such as cycloheximide (CHX) for 2 hour and then exposed to HPB242 for 48 hours. The protein expression of Sp1 was analyzed by western blot analysis. (E) Immunocytochemistry analysis was performed in HPB242 treated HN22 and HSC4 cells. HN22 and HSC4 cells were treated with different concentrations of HPB242 for 48 hours, and cells were immunostained with Sp1 specific antibody, Cleaved-caspase3 specific antibody, and then signals were detected with Jackson 488- and 647-conjugated anti-mouse secondary antibody. DAPI was used for nucleus staining. 254×190 mm (96 × 96 DPI).

Mentions: Several studies have recognized that the expression levels of transcription factor Sp1 dramatically increases during transformation, representing a critical effect in tumor development or maintenance. The effects of HPB242 treatment on Sp1 levels were inspected by western blotting. As shown in Figure 3A, HPB242 treatment led to a sharp decrease in the level of Sp1 in HN22 and HSC4 cells at 48 hours after treatment. In order to characterize the apoptotic action of HPB242, we confirmed expression levels of Cleaved-caspase3 by western blotting (Figure 3B). However, Sp1 mRNA levels did not suppressed by HPB242 in both HN22 and HSC4 cells (Figure 3C). When CHX-pretreated HN22 and HSC4 cells were incubated with HPB242, degradation of Sp1 protein by HPB242 was additionally enhanced (Figure 3D). There were increases in Cleaved-caspase3 following HPB242 (20 μg/ml) treatment of HN22 and HSC4 cells in a time-dependent manner. According to these observations, immunocytochemical results also showed decreased levels of Sp1 positive cells in a dose-dependent manner in HN22 and HSC4 cells (Figure 3E). HPB242 also cell cycle arrest-related proteins such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, Mcl-1 and survivin were significantly decreased (Figure 4A, B). These results collectively suggest that down-regulation of Sp1 by HPB242 treatment could lead to apoptotic cell death.


Specificity protein 1 is a novel target of 2,4-bis (p-hydroxyphenyl)-2-butenal for the suppression of human oral squamous cell carcinoma cell growth.

Chae JI, Lee R, Cho J, Hong J, Shim JH - J. Biomed. Sci. (2014)

The effect of HPB242 on specificity protein 1 (Sp1) protein expression in HN22 and HSC4 cells. HN22 and HSC4 cells were incubated with different concentrations of HPB242 for 48 hours. The cells were harvested and prepared for western blots as described under Methods. Protein expression levels of Sp1 were detected using a specific antibody against Sp1, and its levels were quantified after actin normalization. (A) The HPB242-treated cells were compared with untreated cells, and data are shown as the means ± SD of three independent experiments. The asterisk indicates a significant difference compared with the negative control (untreated cells) (*p < 0.05). (B) Time-dependent effects of HPB242 on Sp1 and Cleaved-caspase3 expression were performed in HN22 and HSC4 cells for 48 hours with 6 hours intervals. (C) The effect of HPB242 (0–20 μg/ml) for 48 hours on Sp1 mRNA expression was determined by RT-PCR. The graphs indicate the ratio of Sp1 to β-actin expression. (D) The effect of HPB242 on Sp1 protein turnover in HN22 and HSC4 cells. The protein lysates were obtained from cells pretreated with protein synthesis inhibitor such as cycloheximide (CHX) for 2 hour and then exposed to HPB242 for 48 hours. The protein expression of Sp1 was analyzed by western blot analysis. (E) Immunocytochemistry analysis was performed in HPB242 treated HN22 and HSC4 cells. HN22 and HSC4 cells were treated with different concentrations of HPB242 for 48 hours, and cells were immunostained with Sp1 specific antibody, Cleaved-caspase3 specific antibody, and then signals were detected with Jackson 488- and 647-conjugated anti-mouse secondary antibody. DAPI was used for nucleus staining. 254×190 mm (96 × 96 DPI).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: The effect of HPB242 on specificity protein 1 (Sp1) protein expression in HN22 and HSC4 cells. HN22 and HSC4 cells were incubated with different concentrations of HPB242 for 48 hours. The cells were harvested and prepared for western blots as described under Methods. Protein expression levels of Sp1 were detected using a specific antibody against Sp1, and its levels were quantified after actin normalization. (A) The HPB242-treated cells were compared with untreated cells, and data are shown as the means ± SD of three independent experiments. The asterisk indicates a significant difference compared with the negative control (untreated cells) (*p < 0.05). (B) Time-dependent effects of HPB242 on Sp1 and Cleaved-caspase3 expression were performed in HN22 and HSC4 cells for 48 hours with 6 hours intervals. (C) The effect of HPB242 (0–20 μg/ml) for 48 hours on Sp1 mRNA expression was determined by RT-PCR. The graphs indicate the ratio of Sp1 to β-actin expression. (D) The effect of HPB242 on Sp1 protein turnover in HN22 and HSC4 cells. The protein lysates were obtained from cells pretreated with protein synthesis inhibitor such as cycloheximide (CHX) for 2 hour and then exposed to HPB242 for 48 hours. The protein expression of Sp1 was analyzed by western blot analysis. (E) Immunocytochemistry analysis was performed in HPB242 treated HN22 and HSC4 cells. HN22 and HSC4 cells were treated with different concentrations of HPB242 for 48 hours, and cells were immunostained with Sp1 specific antibody, Cleaved-caspase3 specific antibody, and then signals were detected with Jackson 488- and 647-conjugated anti-mouse secondary antibody. DAPI was used for nucleus staining. 254×190 mm (96 × 96 DPI).
Mentions: Several studies have recognized that the expression levels of transcription factor Sp1 dramatically increases during transformation, representing a critical effect in tumor development or maintenance. The effects of HPB242 treatment on Sp1 levels were inspected by western blotting. As shown in Figure 3A, HPB242 treatment led to a sharp decrease in the level of Sp1 in HN22 and HSC4 cells at 48 hours after treatment. In order to characterize the apoptotic action of HPB242, we confirmed expression levels of Cleaved-caspase3 by western blotting (Figure 3B). However, Sp1 mRNA levels did not suppressed by HPB242 in both HN22 and HSC4 cells (Figure 3C). When CHX-pretreated HN22 and HSC4 cells were incubated with HPB242, degradation of Sp1 protein by HPB242 was additionally enhanced (Figure 3D). There were increases in Cleaved-caspase3 following HPB242 (20 μg/ml) treatment of HN22 and HSC4 cells in a time-dependent manner. According to these observations, immunocytochemical results also showed decreased levels of Sp1 positive cells in a dose-dependent manner in HN22 and HSC4 cells (Figure 3E). HPB242 also cell cycle arrest-related proteins such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, Mcl-1 and survivin were significantly decreased (Figure 4A, B). These results collectively suggest that down-regulation of Sp1 by HPB242 treatment could lead to apoptotic cell death.

Bottom Line: The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing.HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies.Sp1 was significantly inhibited by HPB242 in a dose-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacy, College of Pharmacy, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam 534-729, Republic of Korea. s1004jh@gmail.com.

ABSTRACT

Background: The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects though 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. The purpose of this study was to investigate the anti-proliferative effects of 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) on two oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, through regulation of specificity protein 1 (Sp1).

Results: HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies. Sp1 was significantly inhibited by HPB242 in a dose-dependent manner. Furthermore, cell cycle regulating proteins and anti-apoptotic proteins, which are known as Sp1 target genes, were altered at the molecular levels. The key important regulators in the cell cycle such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, myeloid leukemia sequence 1 (Mcl-1) and survivin were significantly decreased by HPB242 or suppressed Sp1 levels, however pro-apoptotic proteins caspase3 and PARP were cleaved in HN22 and HSC4.

Conclusions: HPB242 may be useful as a chemotherapeutic agent for OSCC for the purpose of treatment and prevention of oral cancer and for the improvement of clinical outcomes.

Show MeSH
Related in: MedlinePlus