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Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide.

Lin TH, Kuo HC, Chou FP, Lu FJ - BMC Cancer (2008)

Bottom Line: Zymography and Western blot analyses provided information on the effect of As2O3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors.The latter effect was even more pronounced in the presence of 10 microM berberine.The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan. jth.lin@gmail.com

ABSTRACT

Background: Arsenic trioxide (As2O3) exhibits promising anticarcinogenic activity in acute promyelocytic leukemic patients and induces apoptosis in various tumor cells in vitro. Here, we investigated the effect of the natural alkaloid berberine on As2O3-mediated inhibition of cancer cell migration using rat and human glioma cell lines.

Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of rat C6 and human U-87 glioma cells after treatment with As2O3 or berberine, and after co-treatment with As2O3 and berberine. The wound scratch and Boyden chamber assays were applied to determine the effect of As2O3 and berberine on the migration capacity and invasiveness of glioma cancer cells. Zymography and Western blot analyses provided information on the effect of As2O3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors. Most assays were performed three times, independently, and data were analyzed using ANOVA.

Results: The cell viability studies demonstrated that berberine enhances As2O3-mediated inhibition of glioma cell growth after 24 h incubation. Untreated control cells formed a confluent layer, the formation of which was inhibited upon incubation with 5 microM As2O3. The latter effect was even more pronounced in the presence of 10 microM berberine. The As2O3-mediated reduction in motility and invasion of glioma cells was enhanced upon co-treatment with berberine. Furthermore, it has been reported that PKC isoforms influence the morphology of the actin cytoskeleton, as well as the activation of metalloproteases MT1-MMP and MMP-2, reported to be involved in cancer cell migration. Treatment of glioma cells with As2O3 and berberine significantly decreased the activation of PKC alpha and epsilon and led to actin cytoskeleton rearrangements. The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced.

Conclusion: Upon co-treatment of glioma cells with As2O3 and berberine, cancer cell metastasis can be significantly inhibited, most likely by blocking the PKC-mediated signaling pathway involved in cancer cell migration. This study is potentially interesting for the development of novel chemotherapeutic approaches in the treatment of malignant gliomas and cancer development in general.

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Determination of the effects of berberine-As2O3 co-treatment on cell migration and growth using the scratch-wound assay. C6 glioma cells were incubated with 5 μM As2O3 or 10 μM berberine alone and with 5 μM As2O3 and 10 μM berberine for 4, 12, and 24 h and the migration was visualized as described in Methods (A). The percentage of surface area filled by the C6 cells was subsequently quantified by densitometric analyses relative to that of the control which was set at 100% as shown in the graph (B). Data are presented as means ± SD based on three independent experiments. * and ** indicate means that are significantly different when compared to the control group of 12 h incubation with P < 0.05 and P < 0.01, respectively. # and ## indicate means that are significantly different when compared to the control group of 24 h incubation with P < 0.05 and P < 0.01, respectively.
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Figure 2: Determination of the effects of berberine-As2O3 co-treatment on cell migration and growth using the scratch-wound assay. C6 glioma cells were incubated with 5 μM As2O3 or 10 μM berberine alone and with 5 μM As2O3 and 10 μM berberine for 4, 12, and 24 h and the migration was visualized as described in Methods (A). The percentage of surface area filled by the C6 cells was subsequently quantified by densitometric analyses relative to that of the control which was set at 100% as shown in the graph (B). Data are presented as means ± SD based on three independent experiments. * and ** indicate means that are significantly different when compared to the control group of 12 h incubation with P < 0.05 and P < 0.01, respectively. # and ## indicate means that are significantly different when compared to the control group of 24 h incubation with P < 0.05 and P < 0.01, respectively.

Mentions: Data are described as the mean ± standard deviation. For Figure 1, each treatment was compared to its relative control using a Student t test. For Figures 2, 3, 4, and 5, treatment effects were compared using ANOVA with Bonferroni correction. The latter analysis does not apply to Figure 6, but was used to analyze the data presented in Figure 7. The data were analyzed using the SAS statistical software package "SigmaPlot", version 9.0 (SAS Institute Inc., Cary, North Carolina). The significance level was established at P < 0.05. The quantitative data were presented as three repeats from one independent experiment. This is representative of two independent experiments with similar results.


Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide.

Lin TH, Kuo HC, Chou FP, Lu FJ - BMC Cancer (2008)

Determination of the effects of berberine-As2O3 co-treatment on cell migration and growth using the scratch-wound assay. C6 glioma cells were incubated with 5 μM As2O3 or 10 μM berberine alone and with 5 μM As2O3 and 10 μM berberine for 4, 12, and 24 h and the migration was visualized as described in Methods (A). The percentage of surface area filled by the C6 cells was subsequently quantified by densitometric analyses relative to that of the control which was set at 100% as shown in the graph (B). Data are presented as means ± SD based on three independent experiments. * and ** indicate means that are significantly different when compared to the control group of 12 h incubation with P < 0.05 and P < 0.01, respectively. # and ## indicate means that are significantly different when compared to the control group of 24 h incubation with P < 0.05 and P < 0.01, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275285&req=5

Figure 2: Determination of the effects of berberine-As2O3 co-treatment on cell migration and growth using the scratch-wound assay. C6 glioma cells were incubated with 5 μM As2O3 or 10 μM berberine alone and with 5 μM As2O3 and 10 μM berberine for 4, 12, and 24 h and the migration was visualized as described in Methods (A). The percentage of surface area filled by the C6 cells was subsequently quantified by densitometric analyses relative to that of the control which was set at 100% as shown in the graph (B). Data are presented as means ± SD based on three independent experiments. * and ** indicate means that are significantly different when compared to the control group of 12 h incubation with P < 0.05 and P < 0.01, respectively. # and ## indicate means that are significantly different when compared to the control group of 24 h incubation with P < 0.05 and P < 0.01, respectively.
Mentions: Data are described as the mean ± standard deviation. For Figure 1, each treatment was compared to its relative control using a Student t test. For Figures 2, 3, 4, and 5, treatment effects were compared using ANOVA with Bonferroni correction. The latter analysis does not apply to Figure 6, but was used to analyze the data presented in Figure 7. The data were analyzed using the SAS statistical software package "SigmaPlot", version 9.0 (SAS Institute Inc., Cary, North Carolina). The significance level was established at P < 0.05. The quantitative data were presented as three repeats from one independent experiment. This is representative of two independent experiments with similar results.

Bottom Line: Zymography and Western blot analyses provided information on the effect of As2O3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors.The latter effect was even more pronounced in the presence of 10 microM berberine.The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan. jth.lin@gmail.com

ABSTRACT

Background: Arsenic trioxide (As2O3) exhibits promising anticarcinogenic activity in acute promyelocytic leukemic patients and induces apoptosis in various tumor cells in vitro. Here, we investigated the effect of the natural alkaloid berberine on As2O3-mediated inhibition of cancer cell migration using rat and human glioma cell lines.

Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of rat C6 and human U-87 glioma cells after treatment with As2O3 or berberine, and after co-treatment with As2O3 and berberine. The wound scratch and Boyden chamber assays were applied to determine the effect of As2O3 and berberine on the migration capacity and invasiveness of glioma cancer cells. Zymography and Western blot analyses provided information on the effect of As2O3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors. Most assays were performed three times, independently, and data were analyzed using ANOVA.

Results: The cell viability studies demonstrated that berberine enhances As2O3-mediated inhibition of glioma cell growth after 24 h incubation. Untreated control cells formed a confluent layer, the formation of which was inhibited upon incubation with 5 microM As2O3. The latter effect was even more pronounced in the presence of 10 microM berberine. The As2O3-mediated reduction in motility and invasion of glioma cells was enhanced upon co-treatment with berberine. Furthermore, it has been reported that PKC isoforms influence the morphology of the actin cytoskeleton, as well as the activation of metalloproteases MT1-MMP and MMP-2, reported to be involved in cancer cell migration. Treatment of glioma cells with As2O3 and berberine significantly decreased the activation of PKC alpha and epsilon and led to actin cytoskeleton rearrangements. The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced.

Conclusion: Upon co-treatment of glioma cells with As2O3 and berberine, cancer cell metastasis can be significantly inhibited, most likely by blocking the PKC-mediated signaling pathway involved in cancer cell migration. This study is potentially interesting for the development of novel chemotherapeutic approaches in the treatment of malignant gliomas and cancer development in general.

Show MeSH
Related in: MedlinePlus