Use of RNAi silencing to target preconditioned glial cell line-derived neurotrophic factor in neuronal apoptosis

Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may pro-tect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotec-tive effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotro-phic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.

Growth factor receptors: promising drug targets in cancer

Genetic,epigenetic and somatic changes deregulate the expression of growth factor receptors(GFRs),leading to cancer initiation and progression.Tumor cell growth and survival are orchestrated by clonal expansion and evasion of apoptotic signals in cancer cells.The growth of cells is further supported by angiogenesis and metastasis to distant organs.High expression of GFRs also contributes to the development of resistance.Therefore,therapeutics to target GFRs is a potentially attractive molecular approach to treat cancer more effectively.In this review,we have discussed the contribution of GFRs to cancer development and addressed molecular approaches undertaken to inhibit GFR-mediated pathways.A wide number of monoclonal antibodies(mAbs)and protein kinase inhibitors targeting these GFR-mediated functions are in clinical trials to treat human malignancies.However,most drugs that target GFRs lead to the development of drug resistance and generate adverse effects.Nucleic acid-based therapeutics,e.g.short interfering RNA(siRNA)could be harnessed to selectively silence GFR genes in cancer cells.Different polymer,liposome-based nanocarriers,and the most recently developed pH-sensitive inorganic carbonate apatite nanoparticles have been used in cell culture and preclinical trials for cytoplasmic delivery of the siRNAs targeting different GFR genes.siRNA-based therapeutics have been shown to have signifi cant potential to suppress GFR expression and functions and thus could be developed as molecular therapeutics.Multi-targeting of tumors at different levels by combining various approaches along with chemotherapy would be a promising therapeutic approach to fight the disease.Suitable nanocarriers capable of entrapping siRNA,mAb,GFR inhibitors and classical drugs targeting GFR have potential therapeutic applications.

Gene silencing of HIF-2α disrupts glioblastoma stem cell phenotype

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.