Hypoxia Increases Connexin46 and Connexin43 Levels in KNS-42 Glioblastoma Cells

Glioblastoma multiforme is a devastating brain tumor that frequently progresses or recurs despite therapy. We used the glioblastoma-derived cell line, KNS-42, to study the response of the gap junction proteins, connexin46 and connexin43, to chemotherapeutic agents and to prolonged hypoxia to mimic conditions within the tumor microenvironment. Under standard culture conditions, KNS-42 cells have high levels of connexin43 and very low levels of connexin46. The cells that survived temozolomide treatment had increased connexin46 levels and decreased connexin43 levels. In contrast, prolonged hypoxia increased the levels of both connexins, the number of connexin immunopositive cells, and the intensity of the immunofluorescence signal per cell (which localized preferentially in the cytoplasm). Exposure to hypoxia for 12 days decreased the chymotrypsin-like proteasomal activity without altering connexin mRNA levels, suggesting that the changes in connexin levels result from reduced protein degradation. The increased connexin46 in temozolomide-resistant cells suggests that this connexin may have a role in chemotherapy resistance. The results also imply that changes in the microenvironment of glioblastomas (like hypoxia) can alter proteasomal activity and affect levels and subcellular distribution of connexin46 and connexin43. Finally, our data suggest that proteasomal inhibition may not be a good approach to glioblastoma therapy.