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Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library.

Hung MS, Xu Z, Lin YC, Mao JH, Yang CT, Chang PJ, Jablons DM, You L - BMC Cancer (2009)

Bottom Line: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases.In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor.This compound may represent a promising class of CK2 inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA. Ming-Szu.Hung@ucsfmedctr.org

ABSTRACT

Background: Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library.

Methods: We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors in vitro was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays.

Results: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC50 value of 0.55 muM. In addition, hematein inhibited cancer cell growth partially through down-regulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells.

Conclusion: In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors.

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Inhibition effects of hematein on cellular viability and kinase activity in cancer cells. A. A549 cells were treated with serial dilutions of hematein (0 to 200 μM) and cellular viability (normalized to DMSO control) was measured after 48 hours. Data points represent the average of duplicate wells in triplet experiments and bars indicate SD. B. A549 cells were treated with DMSO (control), 50 μM and 100 μM of hematein for 48 hours. Upper western blot panel showed total amount of CK2 used for CK2 kinase assay, and lower table showed relative CK2 kinase activity (normalized to DMSO control) under different hematein concentrations. Data points represent the average of duplicate experiments and bars indicate SD. C. Phosphorylated Akt (Ser 129), total Akt, and PARP were measured by western blot analysis. β-Actin was used as internal loading control. Bands quantization of phosphorylated Akt (Ser 129) was obtained by an analysis with Quantity One 1-D analysis software. Values are reported below each band and normalized to DMSO control. "*" denotes p < 0.05 when compared with control values in triplet experiments. D. The fraction of cells undergoing apoptotic cell death was detected using annexin V FITC and PI stain. Data points represent the average of triplet independent experiments and bars indicate SD. "*" denotes p < 0.05 when compared with the control values.
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Figure 4: Inhibition effects of hematein on cellular viability and kinase activity in cancer cells. A. A549 cells were treated with serial dilutions of hematein (0 to 200 μM) and cellular viability (normalized to DMSO control) was measured after 48 hours. Data points represent the average of duplicate wells in triplet experiments and bars indicate SD. B. A549 cells were treated with DMSO (control), 50 μM and 100 μM of hematein for 48 hours. Upper western blot panel showed total amount of CK2 used for CK2 kinase assay, and lower table showed relative CK2 kinase activity (normalized to DMSO control) under different hematein concentrations. Data points represent the average of duplicate experiments and bars indicate SD. C. Phosphorylated Akt (Ser 129), total Akt, and PARP were measured by western blot analysis. β-Actin was used as internal loading control. Bands quantization of phosphorylated Akt (Ser 129) was obtained by an analysis with Quantity One 1-D analysis software. Values are reported below each band and normalized to DMSO control. "*" denotes p < 0.05 when compared with control values in triplet experiments. D. The fraction of cells undergoing apoptotic cell death was detected using annexin V FITC and PI stain. Data points represent the average of triplet independent experiments and bars indicate SD. "*" denotes p < 0.05 when compared with the control values.

Mentions: Since CK2 kinase showed dose dependent response to hematein inhibition in vitro, we further evaluated inhibition effects of hematein on intact cancer cells. First, A549 lung cancer cells were treated with different concentrations of hematein (0 μM to 200 μM), and cellular viability was measured after 48 hours. Dose dependent response to inhibition of hematein was noted in A549 cells (Fig 4A). We next measured CK2 kinase activity in the lysate of these cells with the same amount of total protein via a radioisotope CK2 kinase assay described in materials and methods section. Dose dependent inhibition responses of CK2 kinase activity were noted in cells treated with 50 μM and 100 μM of hematein (Fig 4B). Interestingly, Akt Ser129, which is phosphorylated by CK2 in vitro and in vivo, also showed significantly decreased phosphorylation in the cells above (Fig 4C). However, total CK2, total Akt and β-actin were comparable. Increased cleaved PARP were also detected in cell lysate treated with 50 μM and 100 μM of hematein (Fig 4C), which indicated increased caspase dependent apoptosis of cancer cells after hematein treatment. Compared to DMSO treated cells, significantly increased apoptotic cells were noted in cells treated with 50 μM and 100 μM of hematein for 48 hours (Fig 4D).


Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library.

Hung MS, Xu Z, Lin YC, Mao JH, Yang CT, Chang PJ, Jablons DM, You L - BMC Cancer (2009)

Inhibition effects of hematein on cellular viability and kinase activity in cancer cells. A. A549 cells were treated with serial dilutions of hematein (0 to 200 μM) and cellular viability (normalized to DMSO control) was measured after 48 hours. Data points represent the average of duplicate wells in triplet experiments and bars indicate SD. B. A549 cells were treated with DMSO (control), 50 μM and 100 μM of hematein for 48 hours. Upper western blot panel showed total amount of CK2 used for CK2 kinase assay, and lower table showed relative CK2 kinase activity (normalized to DMSO control) under different hematein concentrations. Data points represent the average of duplicate experiments and bars indicate SD. C. Phosphorylated Akt (Ser 129), total Akt, and PARP were measured by western blot analysis. β-Actin was used as internal loading control. Bands quantization of phosphorylated Akt (Ser 129) was obtained by an analysis with Quantity One 1-D analysis software. Values are reported below each band and normalized to DMSO control. "*" denotes p < 0.05 when compared with control values in triplet experiments. D. The fraction of cells undergoing apoptotic cell death was detected using annexin V FITC and PI stain. Data points represent the average of triplet independent experiments and bars indicate SD. "*" denotes p < 0.05 when compared with the control values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Inhibition effects of hematein on cellular viability and kinase activity in cancer cells. A. A549 cells were treated with serial dilutions of hematein (0 to 200 μM) and cellular viability (normalized to DMSO control) was measured after 48 hours. Data points represent the average of duplicate wells in triplet experiments and bars indicate SD. B. A549 cells were treated with DMSO (control), 50 μM and 100 μM of hematein for 48 hours. Upper western blot panel showed total amount of CK2 used for CK2 kinase assay, and lower table showed relative CK2 kinase activity (normalized to DMSO control) under different hematein concentrations. Data points represent the average of duplicate experiments and bars indicate SD. C. Phosphorylated Akt (Ser 129), total Akt, and PARP were measured by western blot analysis. β-Actin was used as internal loading control. Bands quantization of phosphorylated Akt (Ser 129) was obtained by an analysis with Quantity One 1-D analysis software. Values are reported below each band and normalized to DMSO control. "*" denotes p < 0.05 when compared with control values in triplet experiments. D. The fraction of cells undergoing apoptotic cell death was detected using annexin V FITC and PI stain. Data points represent the average of triplet independent experiments and bars indicate SD. "*" denotes p < 0.05 when compared with the control values.
Mentions: Since CK2 kinase showed dose dependent response to hematein inhibition in vitro, we further evaluated inhibition effects of hematein on intact cancer cells. First, A549 lung cancer cells were treated with different concentrations of hematein (0 μM to 200 μM), and cellular viability was measured after 48 hours. Dose dependent response to inhibition of hematein was noted in A549 cells (Fig 4A). We next measured CK2 kinase activity in the lysate of these cells with the same amount of total protein via a radioisotope CK2 kinase assay described in materials and methods section. Dose dependent inhibition responses of CK2 kinase activity were noted in cells treated with 50 μM and 100 μM of hematein (Fig 4B). Interestingly, Akt Ser129, which is phosphorylated by CK2 in vitro and in vivo, also showed significantly decreased phosphorylation in the cells above (Fig 4C). However, total CK2, total Akt and β-actin were comparable. Increased cleaved PARP were also detected in cell lysate treated with 50 μM and 100 μM of hematein (Fig 4C), which indicated increased caspase dependent apoptosis of cancer cells after hematein treatment. Compared to DMSO treated cells, significantly increased apoptotic cells were noted in cells treated with 50 μM and 100 μM of hematein for 48 hours (Fig 4D).

Bottom Line: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases.In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor.This compound may represent a promising class of CK2 inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA. Ming-Szu.Hung@ucsfmedctr.org

ABSTRACT

Background: Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library.

Methods: We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors in vitro was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays.

Results: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC50 value of 0.55 muM. In addition, hematein inhibited cancer cell growth partially through down-regulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells.

Conclusion: In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors.

Show MeSH
Related in: MedlinePlus