malignancy and recurrence in glioblastoma multiforme.Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs.Here,we explored the effects of silencing hypoxia inducible fa...malignancy and recurrence in glioblastoma multiforme.Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs.Here,we explored the effects of silencing hypoxia inducible factor-2α(HIF-2α)because of its specificity for CSCs within the hypoxic environment.Methods:Cancer stem cell neurospheres were formed by enriching from both the glioblastoma cell line U87 and from brain tumor stem cells isolated directly from human brain tumors.Silencing of human HIF-2αwas performed using both commercial and in-house transfection of a validated short interfering RNA,with all results compared to an established non-silencing control short interfering RNA.Silencing of HIF-2αwas established by Western blotting,and phenotypic effects were assayed by cell migration assays,cell viability measurements,and immunofluorescence staining of differentiation markers.Results:Transfection with either our previously reported pH-sensitive,cationic amphiphilic macromolecule-based delivery system or Lipofectamine was similarly effective in silencing HIF-2α.The chemotherapeutic resistance and neurosphere formation were reduced when HIF-2αwas silenced.Migratory capacities in the presence of macrophage conditioned media were modulated.HIF-2αsilencing was complementary to temozolomide treatment in producing phenotypic rather than cytotoxic effects.Conclusion:HIF-2αsilencing under hypoxia inhibited CSC phenotypes while promoting differentiated cell phenotypes and is complementary to existing DNA alkylating treatments in inhibiting glioma CSC activity.展开更多
基金This work was supported by a grant from the National Institutes of Health(2R01 EB008278-07).
文摘malignancy and recurrence in glioblastoma multiforme.Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs.Here,we explored the effects of silencing hypoxia inducible factor-2α(HIF-2α)because of its specificity for CSCs within the hypoxic environment.Methods:Cancer stem cell neurospheres were formed by enriching from both the glioblastoma cell line U87 and from brain tumor stem cells isolated directly from human brain tumors.Silencing of human HIF-2αwas performed using both commercial and in-house transfection of a validated short interfering RNA,with all results compared to an established non-silencing control short interfering RNA.Silencing of HIF-2αwas established by Western blotting,and phenotypic effects were assayed by cell migration assays,cell viability measurements,and immunofluorescence staining of differentiation markers.Results:Transfection with either our previously reported pH-sensitive,cationic amphiphilic macromolecule-based delivery system or Lipofectamine was similarly effective in silencing HIF-2α.The chemotherapeutic resistance and neurosphere formation were reduced when HIF-2αwas silenced.Migratory capacities in the presence of macrophage conditioned media were modulated.HIF-2αsilencing was complementary to temozolomide treatment in producing phenotypic rather than cytotoxic effects.Conclusion:HIF-2αsilencing under hypoxia inhibited CSC phenotypes while promoting differentiated cell phenotypes and is complementary to existing DNA alkylating treatments in inhibiting glioma CSC activity.
