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Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo.

Spiller SE, Logsdon NJ, Deckard LA, Sontheimer H - BMC Cancer (2011)

Bottom Line: A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes.We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis.We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Alabama at Birmingham, 35294, USA. sspiller@uab.edu

ABSTRACT

Background: Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer.

Methods: To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition.

Results: We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes 40% smaller than controls.

Conclusions: These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth, and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes.

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Primary tumor expression of NFκB. A-C: Phosphorylated p65 in primary medulloblastoma: immunohistochemical staining of three different primary medulloblastoma tumors with phos(S276)p65 antibody. Magnification: A = 10X; B = 20X; C = 10X. C shows endothelial cell positivity in addition to tumor cells. D: Expression of p65 and phos (S536) p65 in normal brain and tumor in humans [CX = cerebral cortex, CB1 and CB2 = cerebella from 2 different autopsy specimens; mb4, mb2, mb8 = surgical samples of 3 different patient medulloblastoma tumors] along with normal C57Bl/6 mouse cerebellum (CB) compared to Smo/Smo mouse (Smo) cerebellar tumor.
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Figure 4: Primary tumor expression of NFκB. A-C: Phosphorylated p65 in primary medulloblastoma: immunohistochemical staining of three different primary medulloblastoma tumors with phos(S276)p65 antibody. Magnification: A = 10X; B = 20X; C = 10X. C shows endothelial cell positivity in addition to tumor cells. D: Expression of p65 and phos (S536) p65 in normal brain and tumor in humans [CX = cerebral cortex, CB1 and CB2 = cerebella from 2 different autopsy specimens; mb4, mb2, mb8 = surgical samples of 3 different patient medulloblastoma tumors] along with normal C57Bl/6 mouse cerebellum (CB) compared to Smo/Smo mouse (Smo) cerebellar tumor.

Mentions: Primary tumor samples from three cases of medulloblastoma were obtained, and staining with hematoxylin and eosin (H&E) was used to identify the location of tumor cells (not shown). Immunohistochemical (IHC) staining showed that at least 50-90% of evaluable tumor cells strongly express phos(S276) p65, the nuclear transcriptionally active form of p65 (Figure 4A-C). To corroborate this IHC finding, total tissue lysates from 3 more human primary medulloblastoma tumor samples were probed by Western blotting for total p65 and phos(S276)p65. Controls for this assay are human cerebral cortex and cerebellum from adult autopsy tissue. These human tumor samples showed higher levels of total and phosphorylated p65 compared to non-malignant tissue. Additionally, tissue from a brain tumor that developed in a Smo/Smo transgenic medulloblastoma mouse [4] showed an overabundance of p65 and phos(S276) p65 compared to a wild type C57Bl/6 mouse cerebellum (Figure 4D).


Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo.

Spiller SE, Logsdon NJ, Deckard LA, Sontheimer H - BMC Cancer (2011)

Primary tumor expression of NFκB. A-C: Phosphorylated p65 in primary medulloblastoma: immunohistochemical staining of three different primary medulloblastoma tumors with phos(S276)p65 antibody. Magnification: A = 10X; B = 20X; C = 10X. C shows endothelial cell positivity in addition to tumor cells. D: Expression of p65 and phos (S536) p65 in normal brain and tumor in humans [CX = cerebral cortex, CB1 and CB2 = cerebella from 2 different autopsy specimens; mb4, mb2, mb8 = surgical samples of 3 different patient medulloblastoma tumors] along with normal C57Bl/6 mouse cerebellum (CB) compared to Smo/Smo mouse (Smo) cerebellar tumor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094324&req=5

Figure 4: Primary tumor expression of NFκB. A-C: Phosphorylated p65 in primary medulloblastoma: immunohistochemical staining of three different primary medulloblastoma tumors with phos(S276)p65 antibody. Magnification: A = 10X; B = 20X; C = 10X. C shows endothelial cell positivity in addition to tumor cells. D: Expression of p65 and phos (S536) p65 in normal brain and tumor in humans [CX = cerebral cortex, CB1 and CB2 = cerebella from 2 different autopsy specimens; mb4, mb2, mb8 = surgical samples of 3 different patient medulloblastoma tumors] along with normal C57Bl/6 mouse cerebellum (CB) compared to Smo/Smo mouse (Smo) cerebellar tumor.
Mentions: Primary tumor samples from three cases of medulloblastoma were obtained, and staining with hematoxylin and eosin (H&E) was used to identify the location of tumor cells (not shown). Immunohistochemical (IHC) staining showed that at least 50-90% of evaluable tumor cells strongly express phos(S276) p65, the nuclear transcriptionally active form of p65 (Figure 4A-C). To corroborate this IHC finding, total tissue lysates from 3 more human primary medulloblastoma tumor samples were probed by Western blotting for total p65 and phos(S276)p65. Controls for this assay are human cerebral cortex and cerebellum from adult autopsy tissue. These human tumor samples showed higher levels of total and phosphorylated p65 compared to non-malignant tissue. Additionally, tissue from a brain tumor that developed in a Smo/Smo transgenic medulloblastoma mouse [4] showed an overabundance of p65 and phos(S276) p65 compared to a wild type C57Bl/6 mouse cerebellum (Figure 4D).

Bottom Line: A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes.We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis.We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Alabama at Birmingham, 35294, USA. sspiller@uab.edu

ABSTRACT

Background: Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer.

Methods: To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition.

Results: We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes 40% smaller than controls.

Conclusions: These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth, and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes.

Show MeSH
Related in: MedlinePlus