Nrf2/ARE Signaling Directly Regulates SOX9 to Potentially Alter Age-Dependent Cartilage Degeneration.

Oxidative stress is implicated in osteoarthritis, and nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway maintains redox homeostasis. We investigated whether Nrf2/ARE signaling controls SOX9. SOX9 expression in human C-28/I2 chondrocytes was measured by RT-qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap ''n'' collar homology-associated protein 1 (Keap1). To verify whether Nrf2 transcriptionally regulates SOX9, putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL-TK for dual-luciferase assays. SOX9 promoter activities without and with Nrf2-inducer methysticin were compared. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Nrf2-specific RNAi significantly decreased SOX9 expression, whereas Keap1-specific RNAi increased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, but ARE1 excision did not. Functional ARE2 site was essential for methysticin-mediated induction of SOX9 promoter activity. Young Nrf2-knockout mice revealed significantly lower Sox9-positive chondrocytes, and old Nrf2-knockout animals showed thinner cartilage and more cartilage degeneration. Our results suggest Nrf2 directly regulates SOX9 in articular cartilage, and Nrf2-loss can develop mild osteoarthritis at old age. Pharmacological Nrf2 induction may hold the potential to diminish age-dependent cartilage degeneration through improving SOX9 expression.

Study of chemoresistant CD133+ cancer stem cells from human glioblastoma cell line U138MG using multiple assays.

Glioblastoma is one of the most common malignant tumours in adults, with an average life expectancy of less than 1 year. The high mortality of glioblastomas is attributed to its resistance to conventional chemotherapeutic agents. Numerous studies have established the presence of a cancer stem population within glioblastomas. These CSC (cancer stem cell) populations express the cell-surface marker, CD133, and are tumorigenic and chemoresistant. Hence, CSCs make a potential target for anticancer therapies. We have focused on techniques that can reliably identify and isolate a viable CSC population, and studied their chemoresistant attributes. We show the presence of a CSC population within a slowly proliferating glioblastoma cell line, U138MG. An improvised neurosphere enrichment culture technique was developed for the isolation of CSC population. Stem cell neurospheres obtained by this protocol maintained their viability for several weeks, and could be redispersed for deriving colony-forming units and secondary spheres from single-cell suspensions. RT-PCR (reverse transcription-PCR), cell surface localization by immunofluorescence and enumeration by FACS analysis showed that the sphere cultures of U138MG grown on agarose-coated plates had elevated CD133 levels. Drug sensitivity assays indicated that these enriched spheres were more resistant to drug treatment than their non-CSC controls. Drug-resistant CSC had an increased expression of ABC (ATP-binding-cassette) drug transporters, such as ABCC2, ABCC4, ABCG2 and p-glycoprotein, indicative of their role in the resistance mechanisms. These studies will facilitate the development of in vitro assays for the sparse CSC population and strategies for improved treatment regimens for glioblastomas.