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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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Pharmacological inhibition of NFκB in other cells. A: DDTC, PDTC in glioma: Dose response of human U-87MG glioma cells to PDTC and DDTC after 4 days in culture (cell number normalized to control). n = 3 B: PDTC in immature neurons: Dose response of mouse neurosphere stem cells to PDTC (mM): Western analysis probing with anti-cleaved caspase 3(Casp 3) (band ~19kD).
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Figure 2: Pharmacological inhibition of NFκB in other cells. A: DDTC, PDTC in glioma: Dose response of human U-87MG glioma cells to PDTC and DDTC after 4 days in culture (cell number normalized to control). n = 3 B: PDTC in immature neurons: Dose response of mouse neurosphere stem cells to PDTC (mM): Western analysis probing with anti-cleaved caspase 3(Casp 3) (band ~19kD).

Mentions: Interestingly, U-87MG glioma cells treated with DDTC and PDTC were insensitive to both drugs at concentrations <1,000 nM (Figure 2A). Only concentrations >10 μM inhibited U87 proliferation. Thus, medulloblastoma cells are far more sensitive to NFκB inhibition than another malignant brain tumor cell line. There are reports of NFκB constitutive activity in glioma [17,18] and reports to the contrary [19,20]. U-87MG cells are, however, very sensitive to sulfasalazine [20], due to a mechanism of action separate from its NFκB inhibition (discussed below).


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

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

Pharmacological inhibition of NFκB in other cells. A: DDTC, PDTC in glioma: Dose response of human U-87MG glioma cells to PDTC and DDTC after 4 days in culture (cell number normalized to control). n = 3 B: PDTC in immature neurons: Dose response of mouse neurosphere stem cells to PDTC (mM): Western analysis probing with anti-cleaved caspase 3(Casp 3) (band ~19kD).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Pharmacological inhibition of NFκB in other cells. A: DDTC, PDTC in glioma: Dose response of human U-87MG glioma cells to PDTC and DDTC after 4 days in culture (cell number normalized to control). n = 3 B: PDTC in immature neurons: Dose response of mouse neurosphere stem cells to PDTC (mM): Western analysis probing with anti-cleaved caspase 3(Casp 3) (band ~19kD).
Mentions: Interestingly, U-87MG glioma cells treated with DDTC and PDTC were insensitive to both drugs at concentrations <1,000 nM (Figure 2A). Only concentrations >10 μM inhibited U87 proliferation. Thus, medulloblastoma cells are far more sensitive to NFκB inhibition than another malignant brain tumor cell line. There are reports of NFκB constitutive activity in glioma [17,18] and reports to the contrary [19,20]. U-87MG cells are, however, very sensitive to sulfasalazine [20], due to a mechanism of action separate from its NFκB inhibition (discussed below).

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